Mechanistic insights into how the single point mutation change the autoantibody repertoire

Mechanistic insights into how the single point mutation change the autoantibody repertoire | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mechanistic insights into how the single point mutation change the autoantibody repertoire Zhong Ni, Ying Xu, Huimin Zhou, Fangyuan Song, Zhiguo Wang, Dongfeng Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4446391/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2024 Read the published version in The Protein Journal → Version 1 posted 9 You are reading this latest preprint version Abstract A recent study showed that just one point mutation F33 to Y in the H-CDR1 could lead to the autoantibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen ssDNA, and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33 H , W98 H and Y95 L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA, which could be verified by the result that any functional mutation in three key residues (F33 H , W98 H , and Y95 L ) could lead to the loss of ssDNA binding ability of mutant antibody. Our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer. autoantibody molecular dynamic simulation mutation pi-pi stacking repertoire Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Anti-DNA antibodies, including anti-double-stranded(ds) and anti-single-stranded (ss) antibodies, are highly specific markers for systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) patients. Hence, understanding the interaction mechanism between the anti-DNA antibody and DNA is crucial to developing new antibodies for SLE and RA therapy [ 1 ]. For the classical antibody, it is widely accepted that the complementarity determining regions (CDRs), locating on the top of the antibody variable regions, largely contribute to binding with the antigen since they can directly and complementarily contact with the antigen [ 2 ]. Within different CDRs, the H-CDR3 (CDR3 of the antibody heavy chain) was considered as the most critical one for antibody to recognize and bind to antigen [ 3 ]. Previous studies reported that the anti-DNA antibodies bind DNA mainly through electrostatic interactions between the positively charged residues of the antibody CDR and the negatively charged phosphate groups of DNA [ 4 ]. Transferring H-CDR3 from a high-affinity anti-ssDNA antibody to a poor one can render the latter obtain ss-DNA binding ability [ 5 ], which indicates that the H-CDR3 of anti-ds DNA antibodies may have higher proportion of the positive charged residues. Hence, the number of positive charge amino acid in H-CDR3 may largely determine the DNA binding ability of anti-DNA antibody [ 6 ]. However, when the anti-DNA antibody is close to the antigen (DNA), it firstly helps the dsDNA become the ssDNA, then anti-DNA antibody bind with the ssDNA directly [ 7 ], which means that the anti-DNA antibody recognizes and binds antigen not just through electrostatic interactions. Furthermore, more and more data showed that the positive charged amino acid in H-CDR3 contributed less than what we thought [ 8 ]. Therefore, it is postulated that other binding manners exist between anti-DNA antibody and DNA. During the B cell development, the somatic hypermutation (SHM) for V-region of antibody in the germinal center is very important to improve the performance of the antibody. Mutations, even one mutation, introduced in the CDRs through SHM can change the binding properties and repertoire of antibodies [ 9 , 10 ]. However, how just one-point mutation can dramatically change the recognition profiles of the antibody is still unclear. Until now, more and more data about the crystal structures of antibody-DNA complex have been elucidated and speed us to understand the interaction between auto-antibody and DNA, but most of these data are static molecular structures, which cannot display the dynamic recognition and binding process between antibodies and DNA. In addition, the small modification on the fragment antigen-binding (Fab) region of antibody can largely change the binding manner to the antigen [ 11 ], which indicates that it is very difficult to explore the dynamic process of binding between antibody and antigen through conventional laboratory experiments. Therefore, the computer-based molecular dynamics (MD) simulation may be a better approach to perform these “dynamic experiments” at the atomic levels, as it can “revive” the crystallographic structures of protein and provides information not available by other means about their moving parts and intra- and intermolecular interactions [ 12 ]. In this study, we studied the binding behaviors between anti-DNA antibody and DNA using MD simulation. We also used the dysfunctional mutant F33Y that could not bind to DNA to explore how the antibody lost the DNA-binding ability due to one-point mutation. Materials and Methods Protein structure data The original crystal structures of anti-ssDNA antibody binding with and without ssDNA were obtained from the protein data bank (PDB) with the IDs of 5gkr and 5gks [ 7 ], respectively. The crystal structure of its dysfunctional mutant F33Y were also artificially produced by hand, and optimized by Molecular dynamic simulation. Molecular dynamics (MD) simulation MD simulations were performed using the AMBER 19 software. The antibody or antibody-ssDNA complexes were individually immersed into the center of a truncated octahedron box of TIP3P water molecules with a margin distance of 12.0 Å. The environmental potassium counterions were added to keep the system in electrical neutrality. Prior to MD simulations, energy minimizations, including steepest descent and conjugate gradient minimization algorithms, were executed with two steps using the sander program. Initially, only water molecules and counter ions were allowed to move with the restraint energy of 500 kcal/ (mol Å 2 ) on the system (5000 cycles of the steepest descent minimizations, followed by 10,000 cycles of the conjugate gradient minimizations). Next, the whole system was minimized without any restraints (10,000 cycles of the steepest descent minimizations, followed by 20,000 cycles of the conjugate gradient minimizations) After the minimization, each system was heated from 0 to 300 K over 300 ps with the restraint energy of 10 kcal/(mol Å 2 ) on the complex, followed by constant temperature equilibration at 300 K for 1 ns with the same restraint. In the production run, 200 ns NPT MD simulations at constant temperature and pressure (T = 300 K and P = 1 atm) with an integration step of 2 fs were performed to generate trajectories. The SHAKE algorithm was used to restrain all covalent bonds involving hydrogen atoms Principal components analysis (PCA) PCA analysis was carried out for all MD trajectories by using the method of Interactive Essential Dynamics (IED). The PTRAJ module in AmterTools19 was applied for the backbone atoms of antibody or antibody-ssDNA complex in PCA calculation. The graphical summary of motions along the first two eigenvectors was shown in porcupine plot. Non-covalent interactions Non-covalent interactions (NCI) plot calculations were carried out with a step size of 0.10 to visualize the interacting regions between ssDNA and antibodies (wildtype and F33Y mutant) The reduced gradients were rendered as an isosurface in VMD, using an isovalue of 0.3 au. Binding free energy calculations Since the generalized Born (GB) models can make good predictions on the hydration free energy of charged molecules, the binding free energy (ΔG bind ) between hybrid wildtype and/or mutant antibodies and ssDNA were obtained through the MM/GBSA approach. E MM is the gas phase interaction energy comprising internal strain energy (E int ), van der Waals energy (E vdW ) and electrostatic energy (E ele ). G solv is the solvation free energy, including the contributions from a polar part (G GB ) and a nonpolar part (G SA ). ΔE int (bond, angel and dihedral energies) would be cancelled as we used a single trajectory approach to reduce the noise. ΔG GB was estimated using the generalized Born model with the interior and exterior dielectric constants set to 4 and 80, respectively. 500 snapshots were evenly extracted from the last 20 ns trajectories for the calculations of ΔE vdW , ΔE ele , ΔG GB and ΔG SA . Cell culture The CHO cell were cultured in the DMEM medium (Gibco, USA) containing 10% Fetal bovine serum(Gibco, USA), 100ug/mL streptomycin(Sangon Biotech, China), and 100U/mL penicillin G(Sangon Biotech, China)at 37°C in a humidified atmosphere of 5% CO 2 . Antibody gene clone, expression and purification The genes encoding Fab region of the light chain and heavy chains were synthesized by commercial company (Sangon Biotech, China) and subcloned into the reconstituted mammalian expression vectors pcDNA3.1, named as pcDNA-antiDNA-LC and pcDNA-antiDNA-HC, respectively. The plasmids were co-transfected into CHO cells cultured in CHOGrow CD1 serum-free medium (SHANGHAI BASALMEDIA, China) by the Lipofectamine 3000 (Thermofisher, USA) according to manufacturer’s instructions, after 72 hours, the supernatants were harvested and for the next purification. The recombinant antibodies were purified using Protein G beads (Avantorscience, China), In brief, the Protein G resin (1ml) was equilibrated in PBS containing 0.1% NaN 3 , including equilibration, binding, washing steps. The elution in affinity chromatography was operated at the flow rate of 0.4 mL/min. Then 10ml of sample were loaded to the columns, followed by washing with 20 mL PBS containing 0.1% NaN 3 . Bound antibodies were eluted in 20 mL of 50 mM glycine-HCl (pH3.0), and immediately neutralized by addition of 10% volume of 1 M Tris–HCl (pH8.0). Antibody concentrations were determined by BCA protein Assay Kit (Sangon Biotech, China), and the Purity of antibody were estimated by SDS-PAGE. Site-Directed mutagenesis of the anti-DNA antibody Site-directed mutagenesis was performed to produce site-specific mutations at F33L or other positions in anti-DNA antibody. The pcDNA-antiDNA-LC or pcDNA-antiDNA-HC plasmid described above and the mutagenic primers shown in Table 1 (synthesized in Sangon Biotech) were used for this mutagenesis. In brief, PCR amplification was performed with the following 50 µL reaction mixture including 1 µL containing 50 ng of the plasmid was used as the template; 1 µL, containing 125 ng each, of both the forward and reverse primers; 1 µL dNTPs, 5 µL 10×reaction buffer, 0.5 µL KOD polymerase (Toyobo, Japan) and 40 µL of ddH 2 O. One cycle at 95°C for 2 min, followed by 16 cycles in which exposure 95°C for 30 s, 68°C for 8 min were alternated, was used for the PCR amplification. Amplification was followed by add 1uL DpnI (Thermofisher, USA) digestion by incubation at 37°C for 1 h to digest the template DNA. Next, after 10min heating at 80°C, 1 µL of the DpnI (Thermofisher, USA) digest reaction was transformed in to Top10 Competent Cells (Sangon biotech, China) using heat shock at 42°C for 45 s. Cells were then plated on LB agar plates containing 30 µg/mL kanamycin and incubated at 37°C overnight. A single transformed colony was grown in 10 mL of LB medium, containing Ampicillin (Sangon biotech, China) at a concentration of 50 µg/mL, in a shaking incubator at 200 rpm at 37°C overnight. The plasmid was isolated using the mini-prep plasmid isolation kit (Omega, China) and the mutation was confirmed by DNA sequencing in the commercial company (Sangon biotech, China). Table 1 Primers used in this study Mutation site Primers sequences (5' to 3') F33Y F:AGCATCAGCAGC TAC TACTGGAGCTGGATCAGG R:CCTGATCCAGCTCC AGT AGTAGCTGCTGATGCT F33A F:AGCATCAGCAGCTAC GCC TGGAGCTGGATCAGG R:CCTGATCCAGCTCCA GGC GTAGCTGCTGATGCT W98A F:GCCAGGCACAGGAAC GCC CTGTTCGACTACTGG R:CCAGTAGTCGAACAG GGC GTTCCTGTGCCTGGC Y95A F:AGCTACGCCGGCAGC GCC ACCTACGTGTTCGGC R:GCCGAACACGTAGGT GGC GCTGCCGGCGTAGCT Binding ability assay In brief, ELISAs were performed with Maxisorp plates (Thermofisher, USA) coated with 50 µl ssDNA(20-mer T)at a concentration of 10 µg/mL in PBS by overnight incubation. The plate was washed five times with PBS. 50 µl purified antibodies were transferred into the ELISA plate and incubated at 25°C for 2 h. Followed by five times washing with PBST, 100ul of HRP-conjugated anti-human IgG antibody diluted 1/5000 with 2% BSA-PBST was added to each well. The plate was incubated at 25°C for 1 h, followed by washing with PBST. The plate was incubated in the presence of 0.1 mL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Solarbio, China) at 25°C for 15 min. and the reaction was terminated by the addition of 0.1 mL of 0.3 M H 2 SO 4 . Optical densities (OD) were measured at 405 nm using a microplate reader (SYNERGY H4, BioTeK, USA) Results The anti-ssDNA antibody has the innate conformation for binding to the ssDNA The apo (non-binding form) and ssDNA binding forms of anti-ssDNA antibody has been identified by the X-ray crystallography. Their crystal structures, were downed from the protein database bank and with IDs as 5GKR and 5GKS, respectively [ 7 ], which were shown in Fig. 1 A and B. The detailed binding behavior of antibody and ssDNA were analyzed and visualized, as shown in Fig. 1 C, a considerable number of non-bonding interactions between the ssDNA and the antibody were found, e.g. the H-bonds were formed between the amino acid residues Y50, S54, S56, N58, H95 from Heavy chain and the ssDNA, and the Y95 and Y96 from the light chain could form the H-bonds with the ssDNA through their hydroxy groups. In addition, the pi-pi stacking interactions were observed between the aromatic group of side chains of residues and the base groups of the ssDNA, which are Y95 L with the dT2 of the ssDNA, W98 H , F33 L with the dT3 of ssDNA. To explore whether the ssDNA binding affects the arrangement orientation of amino acid residues on the anti-DNA antibody, the key residues derived from the crystal structures of apo (5GKS) or ssDNA binding form (5GKR) were analyzed and the results were shown in Fig. 1 D-F. As shown, the residual orientation between two forms (apo vs ssDNA binding) was consistent and the corresponding RMSD was 0.481 Å, which suggests that the orientation of the key residues would not be influenced by the ssDNA binding. And moreover, the overlapping of CDR loops was also performed to explore the impact on the conformation change of CDR loops due to ssDNA binding, The obtained results showed that no obvious deviation was observed in the CDR loops between apo and ssDNA binding antibody forms (Fig. 1 F). Especially for the H-CDR3, very few or no visible conformation changes were observed after ssDNA binding, take together, these observations suggested that the conformation of this anti-DNA antibody CDR have not been influenced by the binding to antigen ssDNA, implying that this anti-ssDNA antibody has the innate conformation for binding to the ssDNA. Characterize the influence of F33Y mutation on the conformation change of the Variable fragment region It was reported that single-point mutation F33Y in the heavy chain of anti-ssDNA antibody could abolish its ssDNA binding ability [ 7 ]. To investigate the structural change of anti-ssDNA antibody caused by F33Y mutation, 200ns-molecular dynamic simulations procedures were employed. Firstly, the RMSD analysis was performed to evaluate the average amount of movement of backbone atoms throughout the entire protein structure, and the corresponding RMSD values fluctuation were shown in Fig. 2 A. As we can see, the RMSD value of the heavy chain of apo-anti-ssDNA antibody mainly varied between the 1.6Å and 2.8 Å, whereas, the light chain showed relatively high variation of the RMSD value mainly between the 1.8Å and 4Å. In contrast, the RMSD values of the Y33F mutant heavy chain and light chain showed similar variation (between the 1.6Å and 2.8Å). In addition, the superimposition analysis was performed between the crystal structure and the MD-optimized structure, as can be seen in the MD-optimized wildtype anti-ssDNA antibody has only small conformational change relative to the crystal structure (Fig. 2 B). Moreover, the overall architecture of F33Y mutant is similar to the crystal structure of wildtype antibody (Fig. 2 C). These data revealed that F33Y single-point mutation had less or none significant influence on the architecture of this antibody. To assess the conformational flexibility of the wildtype and mutant antibody, the root mean square fluctuations (RMSF) analysis were performed. The RMSF values were calculated by measuring atomic fluctuations after superimposing each structure of a MD trajectory onto the initial structure by means of least-squares fitting, to remove rotational and translational motion [ 13 ]. The results were shown that the corresponding RMSF value variation of amino acids located on the H-CDR1, H-CDR2 and H-CDR3, had the similar trend both in the wildtype and F33Y mutant antibodies, and that the value slightly decreased closed to the 72th residues of F33Y mutant heavy chain (Fig. 2 D), presumably because this region orientated outside of the antibody leading to more flexibility. Interestingly, no obvious difference in the RMSF value was observed in the 33th residues where the mutation F33 to Y happened. However, the RMSF value in the constant 1 region (CH1) of F33Y mutant heavy chain was smaller than that of wildtype, which indicates that the CH1 may be influenced by the H-F33Y mutation although this region is “spatially” far away from the mutation site. For the light chain of the antibody, similar trends were observed that the overall fluctuation of RMSF of the F33Y mutant were similar to those in the wildtype antibody, but some regions were exception, e.g, RMSF values of the resides in the F33Y mutant light chain CDR1 (L-CDR1) was less, whereas the values of L-CDR3 and L-CH1 is larger than those in wildtype. Taken together, these results demonstrated that the F33Y mutation in the heavy chain of anti-ssDNA antibody had more impact on the flexibility/mobility of light chain than heavy chain. PCA helps to determine the most significant motion in dynamics trajectory. It was also carried out to investigate the important motions during the dynamic simulation process, and the covariance matrix of atomic fluctuations was diagonalized for predicting the eigenvalues. As the first few eigenvectors play a central role in the motions of protein, thus the first two eigenvectors of wildtype and F33Y mutant antibody were shown in the Fig. 2 E and F, respectively, as we can see, there exist significant differences in the CDR loops of the light chain, in comparison, less difference existed in the H-CDR loops. But for the constant domains (C H 1-C L ), the motion of the top 2 eigenvectors are quite different between wildtype and F33Y mutant antibodies, it is suggested that F33Y mutation can influence the dynamic behaviors of the CH1 region of antibody. The mutation F33Y impairs the ssDNA binding ability of anti-DNA antibody To further explore the influence of the F33Y mutation on the ssDNA binding of this antibody, the MD simulation analysis was employed to imitate the dynamic process of wildtype and mutant antibody binding to the ssDNA. After 200ns MD simulation, the RMSD of the Cα for the wildtype antibody-ssDNA complex and F33Y mutant antibody-ssDNA complex were detected and collected. After binding to ssDNA, the RMSD values of heavy chains in wild-type antigen-antibody complex was mostly distributed between 2 Å and 3 Å, while the RMSD values of the light chains and the ssDNA in the complex were mostly between 1.6 Å and 2.4 Å and between 0.8 Å and 1.6 Å, respectively (Fig. 3 A). which indicates that the conformation of ssDNA has slightly change in relevant to its initial crystal structure. In contrast, for the F33Y mutant antibody-ssDNA complex, although the RMSD values of the heavy chain were still between 2 Å to 3 Å, both the light chain and ssDNA had increased, especially it reached to 2.2 Å or above for the ssDNA (Fig. 3 B). which indicated that great conformation change happened in ssDNA during its binding to F33Y mutants. To further evaluate the impact of F33Y mutation on antibody heavy and light chains, RMSF analysis were performed. The obtained results were shown in the Fig. 3 C, the superimposition of RMSF plot curves between the F33Ymutant and WT antibodies fitted very well, means that no obvious difference were observed. But for the ssDNA, the RMSF plot of ssDNA in the mutant complex was lower than that in the wildtype complex, which indicated that ssDNA suffered more limitation in the mutant complex. Motivated by these observations, the equilibrated snapshots of wildtype and mutant antibody-ssDNA complexes were collected and compared them with the corresponding initial structures of MD. The superimposition structure was shown in Fig. 3 D and 3 G, obviously, there is no visual differences observed throughout the MD simulation for the WT and mutant complex. But PCA analysis on the dynamic behaviors of the mutant and wildtype antibody showed the CH1 region of the light chain shows quiet different orientation of movement between the WT and F33Y mutant (Fig. 3 E and 3 H). Finally, the binding manner between the ssDNA and antibody were explored and shown in the Fig. 3 F and 3 I, as we can see, when the F33 mutated to Y, the hydroxy group of Y33 can hydrogen-bond with the thymine base of ssDNA (Fig. 3 I), where F33 in the wildtype antibody cannot form the H-bond with the ssDNA (Fig. 3 F). and the dT2 formed more H-bonds in F33Y mutant than in wildtype. Hence, these observations clearly showed that the F33Y mutation could change binding manner between the antibody and ssDNA. F33Y mutation undermine/impair the interaction energy between antibody and ssDNA To investigate the potential interaction mechanism between the ssDNA and antibody based on the energetic changes, the binding free energy of all ssDNA-antibody complexes was evaluated using the MM-GBSA methodology. The predicted binding free energies together with their corresponding energy contributions are summarized in Table 2 . As we can see, the major contribution to stabilize the ssDNA-antibody complex is the electrostatic energy(ΔE ele ), However, comparing the ΔE ele values between the wildtype (-78.4 ± 2.9 kcal/mol) and F33Y mutant (-72.0 ± 4.5kcal/mol) antibodies, it showed that there was no significant difference between them, which suggests that F33Y mutation has weak influence on the electrostatic interactions between the antigen ssDNA and the antibody. The second contributor for the stabilization of ssDNA-antibody complex are van der Waals(vdW) forces (ΔE vdW ), which mainly form non-polar stacking interaction and play a crucial role in the binding between the protein and nucleotide[ 14 , 15 ]. The results showed that ΔE vdW declined from − 54.0 ± 3.6 kcal/mol in wildtype antigen-antibody complex to -41.2 ± 7.1kcal/mol in mutant complex (Table 2 ), which indicates that F33Y mutation in H-CDR1 resulted in significant decrease in vdW interaction. whereas for other energetic items (ΔE GB and ΔE SA ), the difference is less significant in compared with ΔE vdW . So, it can be concluded that the loss of binding ability of F33Y mutant antibody to ssDNA was largely due to the loss the vdW interaction between the antigen ssDNA and F33Y mutant antibody. Table 2 Binding free energies between antibody and ssDNA (kcal∙mol − 1 ) obtained via the MM/GBSA approach for the two models. Antibody Antigen Energy components ΔE ele ΔE vdW ΔE GB ΔE SA ΔE tot WT ssDNA -78.4 ± 2.9 -54.0 ± 3.6 80.7 ± 2.4 -6.6 ± 0.5 -58.3 ± 3.6 F33Y ssDNA -72.0 ± 4.5 -41.2 ± 7.1 74.2 ± 4.5 -5.7 ± 0.5 -44.7 ± 6.9 ΔE tot = ΔE ele + ΔE vdW + ΔE GB + ΔE SA . The stand errors of the mean are listed in parentheses. To further investigate the energy contributions of individual residues, the residue-based free energy decomposition analysis was performed to calculate the energy of residues in the CDR of antibody heavy and light chain. The results showed that the interactions of 23 residues Y32, F33, Y50, I51, Y52, Y53, S54, G55, S56, T57, N58, Y59, K64, R94, H95, R96, N97, W98 in the heavy chain, and Y32, S94, Y95, Y96 in the light chain, were identified as contributor to the binding with the ssDNA (Fig. 4 A, Table 3 ). For the wildtype, the residues F33 H , W98 H , and Y96 L were identified as the top three contributors for the ssDNA binding of the antibody (Fig. 4 A, Table 3 ). For the F33Y mutant antibody-ssDNA complex, the residues Y/F33 H , W98 H , and Y95 L were identified as key contributors. These 23 residues could be divided into four groups according to their properties, 11 amino acids in aromatic groups (11/23), 6 in polar groups (6/23), 3 in charged groups (3/23) and 3 in nonpolar group (3/23) (Table 3 ). Comparing the energy changes among groups, the aromatic groups contribute the most, followed by polar group and charged group, which further demonstrated that the electrostatic interactions does not play a critical role during the antibody-antigen binding process. Table 3 The energy contribution of residues of antibody to its binding with the ssDNA The properties of amino acid Source Amino acid Energy contribution (kcal/mol) WT F33Y Aromatic amino acids H-CDR3 W98 -4.5 -18.36 -4.28 -14.28 L-CDR Y95 -4.22 -2.93 H-CDR1 F/Y33 -3.89 -3.54 H-CDR2 Y52 -3.23 -1.72 H-CDR2 Y50 -1.01 -0.36 L-CDR Y96 -0.43 -0.52 H-CDR3 H95 -0.33 -0.31 H-CDR1 Y32 -0.3 -0.28 H-CDR2 Y53 -0.21 -0.19 L-CDR Y32 -0.14 -0.13 H-FRW3 Y59 -0.1 -0.02 Polar amino acid H-CDR2 S56 -3.34 -8.49 -2.34 -5.6 H-FRW3 T57 -2.06 -0.52 H-CDR3 N97 -1.19 -1.36 H-FRW3 N58 -1.06 -0.2 H-CDR2 S54 -0.73 -1.11 L-CDR S94 -0.11 -0.07 Charged amino acid H-CDR3 R96 -0.45 -0.68 -0.45 -0.63 H-FRW3 K64 -0.13 -0.08 H-CDR3 R94 -0.1 -0.1 Nonpolar amino acid H-CDR2 G55 -0.45 -0.77 -0.69 -0.88 H-CDR2 I51 -0.23 -0.11 H-CDR3 L99 -0.09 -0.08 Furthermore, analyzing the location of these residues found that they were mainly located in the heavy chain CDR1 (H-CDR1, 2/23), CDR2 (H-CDR2, 7/23), CDR3 (H-CDR3, 6/23), framework 3 (H-FRW3, 4/23), and light chain CDRs (L-CDRs, 4/23) (Fig. 4 A). For the wildtype, the H-CDR2 contributed greatest in compared to other regions, followed by the H-CDR3, then L-CDRs, H-CDR1 and H-FRW3 (Fig. 4 B). For the F33Y mutant, H-CDR2 and H-CDR3 have the greatest contributions to the ssDNA binding, followed by H-CDR1 and L-CDRs, then H-FWR3. Yet it is worth noting that the binding energies of H-FR3, H-CDR2, and L-CDRs were largely reduced in F33Y mutant complex comparing with wildtype. Taken together, these results implied that the residues located in these regions might be the most crucial for the binding of antibodies to ssDNA, and that F33Y mutation causing the antibody to lose its antigen binding ability was achieved by changing the energy of the key amino acids in these regions. Pi-pi stacking interaction but not hydrogen bonds between antibody and ssDNA was undermined/impaired upon F33Y mutation Previous study showed that the aromatic pi-pi stacking interaction, a major type of vdW force, was one of the most fundamental interaction forces for the high affinity and specificity of autoantibody [ 16 ]. We identified Y/F33 H , W98 H and Y95 L as key amino acid residues for ssDNA binding in both wild-type and mutant antibody. All of them have aromatic cyclic structures, indicating that they are likely to form stable pi-pi interactions with DNA bases. To verify this, the non-bond interactions detection analyses were performed to inspect the pi-pi stacking interactions between the key residues and the nucleobase of ssDNA. The results showed that F33 H , W98 H and Y95 L could form the stable pi-pi stacking interaction with the nucleotide base group of ssDNA in the wildtype antibody during the whole MD process (Fig. 5 A and 5 B). While for the F33Y mutant, the Y33 H and W98 H could also form the stable pi-pi interaction with the ssDNA, which were similar in the wildtype. However, for the Y95 L in the F33Y mutant, the distance between dT2 and Y95 ( d dT2−−−Y95 ) suddenly increased from the 4 Å to more than 5 Å (critical distance of pi-pi stacking is less than 5 Å) at the end of the MD process, which means that this pi-pi stacking was finally impaired (Fig. 5 C and 5 D). Moreover, it also could see that the dT2 ring of the ssDNA was sandwiched by the F33 and W98 to form the parallel pi-pi-pi stacking interaction in the wildtype antibody-ssDNA complex (Fig. 5 A). However, F33Y mutant, the parallel sandwich pi-pi-pi stacking were destroyed due to that the Y33 cannot form the pi-pi stacking interaction with the dT2 (Fig. 5 C). Hence, these results demonstrated that F33Y mutation weaken the pi-pi stacking interactions between the antibody and ssDNA. Hydrogen bond interaction is also one of the most important non-bonding interactions between the antibody and antigen. To explore whether the hydrogen bond interaction contributes to the ssDNA binding of the wild type and the mutant antibody, the MD simulation timescale was performed. The results showed that Y50 H , T57 H , Y96 H , and W98 H could form the same number of stable hydrogen bonds with ssDNA in both the wild type and the mutant (Table 4 and Fig. 6 ), indicating that the mutation did not alter the way and quantity of these residues binding to DNA. S56 H could form more hydrogen bonds in the wild type, but the N58 H residue could form more stable hydrogen bonds with ssDNA in the F33Y mutant. Additionally, although Y33 H and S54 H in the mutant could form additional hydrogen bonds with ssDNA, these hydrogen bonds had very short duration throughout the entire MD process, implying that these bonds may not be the contributors to the overall stability of the binding process. Table 4 Intermolecular hydrogen bonds between ssDNA and antibody a . Amino acid WT F33Y Acceptor Donor Ocpy b Dist c Ang d Acceptor Donor Ocpy b Dist c Ang d Y50 dT3@O4 Y50 H @HH Y50 H @OH 99.52 2.78 164.43 dT3@O4 Y50 H @HH Y50 H @OH 99.8 2.8 163.9 Y96 dT3@O4 Y96 L @HH Y96 L @OH 98.57 2.82 160.31 dT3@O4 Y96 L @HH Y96 L @OH 98.5 2.8 160.51 W98 dT3@O2 W98 H @H W98 H @N 96.36 2.98 154.36 dT3@O2 W98 H @H W98 H @N 95.4 3 154.09 T57 dT1@O2 T57 H @H T57 H @N 29.79 2.98 147.58 dT1@O2 T57 H @H T57 H @N 53.4 3 142.21 T57 H @O dT2@H3 dT2@N3 32.85 2.95 151.48 T57H@O dT1@H3 dT1@N3 54.7 3.1 146.99 S56 dT2@O4' S56 H @HG S56 H @OG 44.12 2.97 146.9 dT2@O4' S56 H @HG S56 H @OG 56.3 2.9 148.47 dT1@O3' S56 H @HG S56 H @OG 28.95 2.9 144.58 / / / / / N58 dT2@O2 N58 H @HD21 N58 H @ND2 55.67 2.86 162.22 dT2@O2 N58 H @HD21 N58 H @ND2 99.6 2.9 162.42 N58 H @OD1 dT2@H3 dT2@N3 41.74 2.92 162.62 N58 H @OD1 dT2@H3 dT2@N3 96.8 2.9 161.86 / / / / / N58 H @OD1 dT1@H3 dT1@N3 28.9 3.1 136.85 Y33 / / / / / dT2@O3' Y33 H @ HH Y33 H @ OH 36.7 3 138.13 / / / / / dT2@O2 Y33 H @ HH Y33 H @ OH 35.8 2.9 146.07 S54 / / / / / dT2@OP1 S54 H @HG S54 H @OG 45.8 2.7 163.57 a The subscripted H and L indicate the residues reside in the antibody heavy and light chains, respectively. b Hydrogen bond occupancy during MD (%). c Time averaged hydrogen bond length (Å). d Time averaged hydrogen bond angle (º). Pi-pi stacking interactions dominated the binding between antibody and ssDNA In order to confirm the dominated role of the pi-pi stacking interaction between the anti-DNA antibody and the ssDNA, the F33Y H , F33A H , W98A H , Y95A L mutants and the wildtype antibody were expressed and purified (Fig. 7 A). Their binding performance towards the ssDNA were evaluated by the ELISA experiments. The corresponding results showed that almost all the mutants lost the ssDNA binding ability (Fig. 7 B) due to impairing of pi-pi stacking interaction, which further demonstrated that pi-pi stacking was one of the key contributors for this anti-ssDNA antibody. Discussion During the recognition and binding process of antibodies toward antigen, each portion of the antibody variable region plays different effects in the identification and binding antigen molecules, especially the CDR3 on the heavy chain, which is recombinantly recombined by the VDJ encoding an antibody gene. It is believed that the H-CDR3 of the antibody plays a decisive role in identifying antigen and binding antigen. So in this study, MD simulation results showed that there are less/none influence on the contribution of H-CDR3 to binding with ssDNA upon introducing mutation F33Y in the H-CDR1, And together with the analysis on the crystal structures of the complex (antibody-ssDNA complex) and apo-antibody which showed that no obvious difference in the conformation of the CDRs were observed upon on the binding with ssDNA, indicating that the antibody may have a tendency to be born with binding to ssDNA. According to our finding, the contribution of different regions on binding to ssDNA is in this order: H-CDR2 > H-CDR3 > L-CDRs > or = H-CDR1 > H-FRW3, that means that H-CDR2 also play similar important role in binding with the antigen ssDNA as compared with the H-CDR3. H-FRW3 was traditionally considered as the scaffold to support the CDRs, but it is found to interact with antigen directly in this study. This may be the particular characteristics of this anti-ssDNA antibody, and anti-DNA antibodies may take other ways to achieve identification and binding of ssDNA. When the H-F33 mutate to Y, the contribution from the HCDR2 and H-FRW3 of F33Y mutant were significantly decreased to almost half of that in the wildtype. But the other regions were not largely influenced by this mutation. These results can at least partly explain that why F33Y mutant lost the binding ability toward the antigen ssDNA. Additionally, the function of H-FRW3 was need to be recognized since it was found to interact with antigen directly, which was traditionally considered just as the scaffold to support the CDRs. This results is consisted with the Victor Ovchinnikov’s findings, that high frequency mutation in the H-FRW3 can expand diversity of anti-HIV neutralizing antibody [ 17 ], implied that the H-FRW3 not only act as the scaffold to support the CDRs, but also affect the binding properties of CDR toward the antigen. And in this study, the mutation (F33Y) located in the H-CDR1, which is spatially separated from the H-FRW3, but this mutation significant influence the binding of H-FRW3 to the antigen ssDNA possible through the allosteric effect. Previous studies pointed out the CH1 domain influences the antibody paratope and in turn its specificity and functional activities [ 18 ] and, Lavoie et.al found that the Fv–HEL complex was found to have a dissociation constant one order of magnitude lower than that of the Fab–HEL complex [ 19 ]. Moreover, CH1 loops implicated in interactions between H and L chains which show the most consistent and substantial changes upon Ag binding [ 20 ] in this study, we found that the mutation (F33Y) located in the H-CDR1 can influence the dynamic behavior of the CH1 region of the Fab. PCA analysis on the MD trajector clearly showed that the movement of CH1 showed quiet different behavior upon inducing F33Y mutation, which further verified that the C and V regions are structurally coupled and affect each other mainly by the Allosteric effects, So this study can give us hint in antibody humanized engineering, just graft the CDRs may lead to lost its original ability of antibody. In previous years, Tanner and co-workers identified a conserved and fundamental structural element responsible for the recognition of ssDNA, termed the “ssDNA-antibody recognition module” (D-ARM) [ 21 ]. The D-ARM consists of a tyrosine residue that stacks with the base and a glycine that forms a hydrogen bond with the base. Thus, the H-bond interaction and aromatic group stacking interaction are the main contributor for the autoantibody binding to its antigen. In present study, it was found that F33Y mutation in H-CDR1 can eliminate the ssDNA binding ability of this antibody, the H-bonding interaction between the mutant antibody and ssDNA were not weaken in compared with the wildtype antibody, but the pi-pi stacking interaction derived from the aromatic residues and base groups of DNA were significantly impaired upon the F33Y mutation, also the experimental results showed that any one mutation in three key residues (F33 H , W98 H , and Y95 L ) lead to the loss of ssDNA binding ability of mutant antibody. these findings at least partly indicated that pi-pi stacking interaction between the antibody and antigen is one of major factor that determine the whether the antibody can bind with DNA. These results are consistent with previous study that the three Aromatic Residues W56, W75 and F79 form a stable pi-pi stacking effect for ssDNA-binding Protein [ 22 ]. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This study was supported by grants from the Jiangsu University, China (18JDG028). Author Contributions Statement Z.N. and D.C. analyzed the data, prepared figures 1- 6 and wrote the main manuscript. Y.X., H.Z. and F. S. collected the data and prepared figures 7 and tables 1-4. Z.W. contributed to scientific discussion and critical review of the manuscript. Z.N. and D.C. conceived the idea and designed the structures of the paper. All authors contributed to the article and approved the submitted version. References McInnes IB, Schett G (2011) The Pathogenesis of Rheumatoid Arthritis. New Engl J Med 365: 2205-2219. doi:/ 10.1056/NEJMra1004965 van Regenmortel MH (2000) The recognition of proteins and peptides by antibodies. J Immunoassay 21: 85-108. doi:/10.1080/01971520009349530 Leal E, Jaloma-Cruz ARBarros-Nunez P (2004) High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemia. Hematol Oncol 22: 55-61. doi:/10.1002/hon.727 Kalinina O, Doyle-Cooper CM, Miksanek J, et al. (2011) Alternative mechanisms of receptor editing in autoreactive B cells. Proc Natl Acad Sci U S A 108: 7125-30. doi:/10.1073/pnas.1019389108 Komissarov AA, Calcutt MJ, Marchbank MT, et al. (1996) Equilibrium binding studies of recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining regions. J Biol Chem 271: 12241-6. doi:/10.1074/jbc.271.21.12241 Jang YJStollar BD (2003) Anti-DNA antibodies: aspects of structure and pathogenicity. Cell Mol Life Sci 60: 309-20. doi:/10.1007/s000180300026 Sakakibara S, Arimori T, Yamashita K, et al. (2017) Clonal evolution and antigen recognition of anti-nuclear antibodies in acute systemic lupus erythematosus. Sci Rep 7: 16428. doi:/10.1038/s41598-017-16681-y Beckingham JA, Cleary J, Bobeck M, et al. (2003) Kinetic analysis of sequence-specific recognition of ssDNA by an autoantibody. Biochemistry 42: 4118-26. doi:/10.1021/bi020658k Tsumoto KKumagai I (2000) Thermodynamic consequences of single-mutation on association of an antibody with its specific antigen: The case of HyHEL-10-hen lysozyme complex. Chemistry Letters: 1066-1067. doi:/DOI 10.1246/cl.2000.1066 Saul FA, Vulliez-Le Normand B, Passafiume M, et al. (2000) Structure of the Fab fragment from F124, a monoclonal antibody specific for hepatitis B surface antigen. Acta Crystallogr D Biol Crystallogr 56: 945-51. doi:/10.1107/s0907444900008088 Sinha N, Li Y, Lipschultz CA, et al. (2007) Understanding antibody-antigen associations by molecular dynamics simulations: detection of important intra- and inter-molecular salt bridges. Cell Biochem Biophys 47: 361-75. doi:/10.1007/s12013-007-0031-8 Turonova B, Sikora M, Schurmann C, et al. (2020) In situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hinges. Science 370: 203-208. doi:/10.1126/science.abd5223 Wilson KA, Holland DJWetmore SD (2016) Topology of RNA-protein nucleobase-amino acid pi-pi interactions and comparison to analogous DNA-protein pi-pi contacts. RNA 22: 696-708. doi:/10.1261/rna.054924.115 Bobeck MJ, Rueda D, Walter NG, et al. (2007) Structural modeling of sequence specificity by an autolantibody against single-stranded DNA. Biochemistry 46: 6753-6765. doi:/10.1021/bi700212s Ovchinnikov V, Louveau JE, Barton JP, et al. (2018) Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies. Elife 7. doi:/10.7554/eLife.33038 Lavoie TB, Drohan WNSmith-Gill SJ (1992) Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. J Immunol 148: 503-13 Sela-Culang I, Kunik VOfran Y (2013) The structural basis of antibody-antigen recognition. Front Immunol 4: 302. doi:/10.3389/fimmu.2013.00302 Tanner JJ, Komissarov AADeutscher SL (2001) Crystal structure of an antigen-binding fragment bound to single-stranded DNA. J Mol Biol 314: 807-22. doi:/10.1006/jmbi.2001.5178 Gamsjaeger R, Kariawasam R, Gimenez AX, et al. (2015) The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus. Biochem J 465: 337-46. doi:/10.1042/BJ20141140 Khamassi M, Xu L, Rey J, et al. (2020) The CH1alpha domain of mucosal gp41 IgA contributes to antibody specificity and antiviral functions in HIV-1 highly exposed Sero-Negative individuals. PLoS Pathog 16: e1009103. doi:/10.1371/journal.ppat.1009103 Li J, Wang Y, An L, et al. (2018) Direct Observation of CH/CH van der Waals Interactions in Proteins by NMR. J Am Chem Soc 140: 3194-3197. doi:/10.1021/jacs.7b13345 Victora GDNussenzweig MC (2022) Germinal Centers. Annu Rev Immunol 40: 413-442. doi:/10.1146/annurev-immunol-120419-022408 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2024 Read the published version in The Protein Journal → Version 1 posted Editorial decision: Revision requested 30 May, 2024 Reviews received at journal 30 May, 2024 Reviews received at journal 28 May, 2024 Reviewers agreed at journal 22 May, 2024 Reviewers agreed at journal 22 May, 2024 Reviewers invited by journal 22 May, 2024 Editor assigned by journal 22 May, 2024 Submission checks completed at journal 22 May, 2024 First submitted to journal 19 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4446391","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":308712371,"identity":"9e35e3cf-eb70-4fad-b393-c0bca4ab93c4","order_by":0,"name":"Zhong Ni","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Zhong","middleName":"","lastName":"Ni","suffix":""},{"id":308712372,"identity":"10e712f8-0318-4792-8dc1-e7b3c109b125","order_by":1,"name":"Ying Xu","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Xu","suffix":""},{"id":308712373,"identity":"ae57bee4-a100-4dae-89f0-1750697545c0","order_by":2,"name":"Huimin Zhou","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Huimin","middleName":"","lastName":"Zhou","suffix":""},{"id":308712374,"identity":"d9e28d35-3370-4d4f-b3f4-4d51e00dfcf4","order_by":3,"name":"Fangyuan Song","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Fangyuan","middleName":"","lastName":"Song","suffix":""},{"id":308712375,"identity":"c597f9e0-ff8d-413a-a089-e188d9c73f4c","order_by":4,"name":"Zhiguo Wang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Zhiguo","middleName":"","lastName":"Wang","suffix":""},{"id":308712376,"identity":"e44ca093-57d6-4489-b754-f2613280f951","order_by":5,"name":"Dongfeng Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIie3RMYvCMBTA8VeEdim6psN9h0CgCAX9Kq8E6lKcOzhkqoty630PwTkSqEt07nCDIIhj7gvIPfwANbcdmD+8hMD7TQEIhf5jmqahmQAgXSNPYmky9WfC9fPpQcbHrbnr5nu26/HqoClKlZz0IMnsuZpqe5P7HisGdlGqdImDhPd1Ln5aI3MiELWmVCzlLwk/PIwUXyhd9PAj4nJQZsYZIouUB8mszUF3Bpm9VAy7hWjTepiMjxvh9MrMJ+taOrcqPj4TO0yomNFRKkjx+Znxq31q5OiYAyTaYzkUCoXesV9Ed1DRsTE5CgAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangsu University","correspondingAuthor":true,"prefix":"","firstName":"Dongfeng","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-05-20 03:38:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4446391/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4446391/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10930-024-10225-w","type":"published","date":"2024-07-28T04:02:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57646694,"identity":"ac33b689-816d-427a-9a9b-cb9011d65d9f","added_by":"auto","created_at":"2024-06-03 19:49:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":826919,"visible":true,"origin":"","legend":"\u003cp\u003eThe conformation difference between the apo and ssDNA-binding antibody.\u003c/p\u003e\n\u003cp\u003eA) The apo form of the anti-DNA antibody;\u003c/p\u003e\n\u003cp\u003eB) The ssDNA-binding form of the anti-DNA antibody;\u003c/p\u003e\n\u003cp\u003eC) The binding manner between this antibody to ssDNA;\u003c/p\u003e\n\u003cp\u003eD) The key residues that interacted with the ssDNA;\u003c/p\u003e\n\u003cp\u003eE) The overlay of the key residues between the apo and ssDNA-binding forms of antibody;\u003c/p\u003e\n\u003cp\u003eF) The overlay of the CDRs loop between the apo and ssDNA-binding forms of antibody.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/fcc534e6ae219df8803f5eeb.jpg"},{"id":57646695,"identity":"da72a69b-cdb3-430e-921c-73fc44360ca1","added_by":"auto","created_at":"2024-06-03 19:49:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5141603,"visible":true,"origin":"","legend":"\u003cp\u003eThe dynamics analysis on the wildtype and F33Y mutant anti-DNA antibodies during MD simulation.\u003c/p\u003e\n\u003cp\u003eA) Root mean square deviation (RMSD) plot for Fab of wildtype and F33Y mutant anti-DNA antibody;\u003c/p\u003e\n\u003cp\u003eB) the superimposition of equilibrated snapshot of the wildtype antibody with its crystal structure;\u003c/p\u003e\n\u003cp\u003eC) the superimposition of equilibrated snapshot of the F33Y mutant antibody with its initial structure derived from MD simulation;\u003c/p\u003e\n\u003cp\u003eD) Root mean square fluctuation (RMSF) plot for Fabs of wildtype and F33Y mutant antibody;\u003c/p\u003e\n\u003cp\u003eE) and F) Porcupine plot to visualize the first and second principle eigenvectors of the wildtype (E) and F33Y mutant (F) anti-DNA antibody. The arrow attached to the Cα atoms show the direction of motion and the length of arrows shows the magnitude of the eigenvector\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/d2ed1c1dc4db61569ba9b04a.jpg"},{"id":57646696,"identity":"51be1d21-32a4-4da2-a28d-421c0a5c9d8b","added_by":"auto","created_at":"2024-06-03 19:49:26","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6601387,"visible":true,"origin":"","legend":"\u003cp\u003eThe dynamics of wildtype and F33Y mutant anti-DNA antibodies or their complex with the ssDNA during MD simulation.\u003c/p\u003e\n\u003cp\u003eA) Root mean square deviation (RMSD) plot for Fab of wildtype anti-DNA antibody and its antigen ssDNA;\u003c/p\u003e\n\u003cp\u003eB) Root mean square deviation plot the Fab of F33Y mutant antibody and its antigen ssDNA;\u003c/p\u003e\n\u003cp\u003eC) Root mean square fluctuation (RMSF) plot for Fabs of wildtype and F33Y mutant antibody and ssDNA;\u003c/p\u003e\n\u003cp\u003eD) Superimposed comparison between the initial and the equilibrated snapshot of the wildtype anti-DNA antibody-ssDNA complexes;\u003c/p\u003e\n\u003cp\u003eE) Porcupine plot to visualize the first and second principle eigenvectors of the wildtype anti-DNA antibody. The arrow attached to the Cα atoms show the direction of motion and the length of arrows shows the magnitude of the eigenvector;\u003c/p\u003e\n\u003cp\u003eF) Close views of the binding site of wildtype antibody-ssDNA complex. The pink sticks represent the key residues in the light chain, the cyan sticks represent key residues in the heavy chain, the violet sticks represent ssDNA. Yellow dotted lines represent the H-bonding interaction;\u003c/p\u003e\n\u003cp\u003eG) Superimposed comparison between the initial and the equilibrated snapshot of the F33Y mutant anti-DNA antibody-ssDNA complexes;\u003c/p\u003e\n\u003cp\u003eH) Porcupine plot to visualize the first and second principle eigenvectors of the wildtype anti-DNA antibody. The arrow attached to the Cα atoms show the direction of motion and the length of arrows shows the magnitude of the eigenvector;\u003c/p\u003e\n\u003cp\u003eI) Close views of the binding site of F33Y mutant antibody-ssDNA complex. The pink sticks represent the key residues in the light chain, the cyan sticks represent key residues in the heavy chain, the violet sticks represent ssDNA. Yellow dotted lines represent the H-bonding interaction\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/09f8f3081de69d7528708553.jpg"},{"id":57647105,"identity":"bb2a355c-2449-4622-a612-891b3aa76e71","added_by":"auto","created_at":"2024-06-03 19:57:26","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":264888,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis on the corresponding energetic contribution of key residues of the antibody to the binding toward the antigen ssDNA.\u003c/p\u003e\n\u003cp\u003eA) The comparison in Energetic Contribution of the key residues to the binding with ssDNA between the wildtype and F33Y mutant antibodies.\u003c/p\u003e\n\u003cp\u003eB) \u0026nbsp;The comparison in Energetic Contribution of the key regions to the binding with ssDNA between the wildtype and F33Y mutant antibodies\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/87e1d36e94f1b0b9015b188f.jpg"},{"id":57647106,"identity":"7bc377f8-2e26-494b-9516-4eca929b1b8f","added_by":"auto","created_at":"2024-06-03 19:57:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1842935,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis on the non-bonding interaction between the ssDNA and antibodies\u003c/p\u003e\n\u003cp\u003eA) the nonbonding interaction between the ssDNA and wildtype antibody;\u003c/p\u003e\n\u003cp\u003eB) the corresponding aromatic ring distance between the residues and nucleotide acid;\u003c/p\u003e\n\u003cp\u003eC) the nonbonding interaction between the ssDNA and F33Y mutant antibody;\u003c/p\u003e\n\u003cp\u003eD) the corresponding aromatic ring distance between the residues and nucleotide acid.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/cabd3f15ecb5ec72f1efcdf9.jpg"},{"id":57646701,"identity":"33b7e336-dcd1-4042-9c6b-fd4aaebb239d","added_by":"auto","created_at":"2024-06-03 19:49:26","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":826029,"visible":true,"origin":"","legend":"\u003cp\u003eH-bonding interaction between the antibodies (Wildtype or F33Ymutant) and antigen ssDNA.\u003c/p\u003e\n\u003cp\u003eA), B) and C) The deviation of distance between h-bond donor and H-bond acceptor in the ssDNA-antibody complex;\u003c/p\u003e\n\u003cp\u003eD), E), F), and G) The deviation of the deviation of distance between h-bond donor and H-bond acceptor in the ssDNA- mutant antibody complex\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/c456b7632e29e919400e8a77.jpg"},{"id":57646699,"identity":"1d51d9a9-ed65-4d07-bd98-ae799234391e","added_by":"auto","created_at":"2024-06-03 19:49:26","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":388911,"visible":true,"origin":"","legend":"\u003cp\u003eExperiments verified the impact of the mutation on antibody binding ability\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA) SDS-PAGE analysis of purified antibody and its mutants;\u003c/p\u003e\n\u003cp\u003eB) ELISA assay on the binding of antibodies toward the ssDNA. Averages of triplicate measurements are shown with s.e.m.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/de00f67491f02a12cb293108.jpg"},{"id":61374790,"identity":"b6925040-adb4-4969-bff8-371847bc7b2b","added_by":"auto","created_at":"2024-07-30 04:02:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16773450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4446391/v1/7dd35c3f-7e69-46b4-9f0e-5c0fca90f9d6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanistic insights into how the single point mutation change the autoantibody repertoire","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnti-DNA antibodies, including anti-double-stranded(ds) and anti-single-stranded (ss) antibodies, are highly specific markers for systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) patients. Hence, understanding the interaction mechanism between the anti-DNA antibody and DNA is crucial to developing new antibodies for SLE and RA therapy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For the classical antibody, it is widely accepted that the complementarity determining regions (CDRs), locating on the top of the antibody variable regions, largely contribute to binding with the antigen since they can directly and complementarily contact with the antigen [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Within different CDRs, the H-CDR3 (CDR3 of the antibody heavy chain) was considered as the most critical one for antibody to recognize and bind to antigen [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Previous studies reported that the anti-DNA antibodies bind DNA mainly through electrostatic interactions between the positively charged residues of the antibody CDR and the negatively charged phosphate groups of DNA [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Transferring H-CDR3 from a high-affinity anti-ssDNA antibody to a poor one can render the latter obtain ss-DNA binding ability [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], which indicates that the H-CDR3 of anti-ds DNA antibodies may have higher proportion of the positive charged residues. Hence, the number of positive charge amino acid in H-CDR3 may largely determine the DNA binding ability of anti-DNA antibody [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, when the anti-DNA antibody is close to the antigen (DNA), it firstly helps the dsDNA become the ssDNA, then anti-DNA antibody bind with the ssDNA directly [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which means that the anti-DNA antibody recognizes and binds antigen not just through electrostatic interactions. Furthermore, more and more data showed that the positive charged amino acid in H-CDR3 contributed less than what we thought [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, it is postulated that other binding manners exist between anti-DNA antibody and DNA.\u003c/p\u003e \u003cp\u003eDuring the B cell development, the somatic hypermutation (SHM) for V-region of antibody in the germinal center is very important to improve the performance of the antibody. Mutations, even one mutation, introduced in the CDRs through SHM can change the binding properties and repertoire of antibodies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, how just one-point mutation can dramatically change the recognition profiles of the antibody is still unclear.\u003c/p\u003e \u003cp\u003eUntil now, more and more data about the crystal structures of antibody-DNA complex have been elucidated and speed us to understand the interaction between auto-antibody and DNA, but most of these data are static molecular structures, which cannot display the dynamic recognition and binding process between antibodies and DNA. In addition, the small modification on the fragment antigen-binding (Fab) region of antibody can largely change the binding manner to the antigen [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which indicates that it is very difficult to explore the dynamic process of binding between antibody and antigen through conventional laboratory experiments. Therefore, the computer-based molecular dynamics (MD) simulation may be a better approach to perform these \u0026ldquo;dynamic experiments\u0026rdquo; at the atomic levels, as it can \u0026ldquo;revive\u0026rdquo; the crystallographic structures of protein and provides information not available by other means about their moving parts and intra- and intermolecular interactions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we studied the binding behaviors between anti-DNA antibody and DNA using MD simulation. We also used the dysfunctional mutant F33Y that could not bind to DNA to explore how the antibody lost the DNA-binding ability due to one-point mutation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtein structure data\u003c/h2\u003e \u003cp\u003eThe original crystal structures of anti-ssDNA antibody binding with and without ssDNA were obtained from the protein data bank (PDB) with the IDs of 5gkr and 5gks [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], respectively. The crystal structure of its dysfunctional mutant F33Y were also artificially produced by hand, and optimized by Molecular dynamic simulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMolecular dynamics (MD) simulation\u003c/h2\u003e \u003cp\u003eMD simulations were performed using the AMBER 19 software. The antibody or antibody-ssDNA complexes were individually immersed into the center of a truncated octahedron box of TIP3P water molecules with a margin distance of 12.0 \u0026Aring;. The environmental potassium counterions were added to keep the system in electrical neutrality.\u003c/p\u003e \u003cp\u003ePrior to MD simulations, energy minimizations, including steepest descent and conjugate gradient minimization algorithms, were executed with two steps using the sander program. Initially, only water molecules and counter ions were allowed to move with the restraint energy of 500 kcal/ (mol \u0026Aring;\u003csup\u003e2\u003c/sup\u003e) on the system (5000 cycles of the steepest descent minimizations, followed by 10,000 cycles of the conjugate gradient minimizations). Next, the whole system was minimized without any restraints (10,000 cycles of the steepest descent minimizations, followed by 20,000 cycles of the conjugate gradient minimizations)\u003c/p\u003e \u003cp\u003eAfter the minimization, each system was heated from 0 to 300 K over 300 ps with the restraint energy of 10 kcal/(mol \u0026Aring;\u003csup\u003e2\u003c/sup\u003e) on the complex, followed by constant temperature equilibration at 300 K for 1 ns with the same restraint. In the production run, 200 ns NPT MD simulations at constant temperature and pressure (T\u0026thinsp;=\u0026thinsp;300 K and P\u0026thinsp;=\u0026thinsp;1 atm) with an integration step of 2 fs were performed to generate trajectories. The SHAKE algorithm was used to restrain all covalent bonds involving hydrogen atoms\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePrincipal components analysis (PCA)\u003c/h2\u003e \u003cp\u003ePCA analysis was carried out for all MD trajectories by using the method of Interactive Essential Dynamics (IED). The PTRAJ module in AmterTools19 was applied for the backbone atoms of antibody or antibody-ssDNA complex in PCA calculation. The graphical summary of motions along the first two eigenvectors was shown in porcupine plot.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eNon-covalent interactions\u003c/h2\u003e \u003cp\u003eNon-covalent interactions (NCI) plot calculations were carried out with a step size of 0.10 to visualize the interacting regions between ssDNA and antibodies (wildtype and F33Y mutant) The reduced gradients were rendered as an isosurface in VMD, using an isovalue of 0.3 au.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBinding free energy calculations\u003c/h2\u003e \u003cp\u003eSince the generalized Born (GB) models can make good predictions on the hydration free energy of charged molecules, the binding free energy (ΔG\u003csub\u003ebind\u003c/sub\u003e) between hybrid wildtype and/or mutant antibodies and ssDNA were obtained through the MM/GBSA approach. E\u003csub\u003eMM\u003c/sub\u003e is the gas phase interaction energy comprising internal strain energy (E\u003csub\u003eint\u003c/sub\u003e), van der Waals energy (E\u003csub\u003evdW\u003c/sub\u003e) and electrostatic energy (E\u003csub\u003eele\u003c/sub\u003e). G\u003csub\u003esolv\u003c/sub\u003e is the solvation free energy, including the contributions from a polar part (G\u003csub\u003eGB\u003c/sub\u003e) and a nonpolar part (G\u003csub\u003eSA\u003c/sub\u003e). ΔE\u003csub\u003eint\u003c/sub\u003e (bond, angel and dihedral energies) would be cancelled as we used a single trajectory approach to reduce the noise. ΔG\u003csub\u003eGB\u003c/sub\u003e was estimated using the generalized Born model with the interior and exterior dielectric constants set to 4 and 80, respectively. 500 snapshots were evenly extracted from the last 20 ns trajectories for the calculations of ΔE\u003csub\u003evdW\u003c/sub\u003e, ΔE\u003csub\u003eele\u003c/sub\u003e, ΔG\u003csub\u003eGB\u003c/sub\u003e and ΔG\u003csub\u003eSA\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe CHO cell were cultured in the DMEM medium (Gibco, USA) containing 10% Fetal bovine serum(Gibco, USA), 100ug/mL streptomycin(Sangon Biotech, China), and 100U/mL penicillin G(Sangon Biotech, China)at 37\u0026deg;C in a humidified atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntibody gene clone, expression and purification\u003c/h2\u003e \u003cp\u003eThe genes encoding Fab region of the light chain and heavy chains were synthesized by commercial company (Sangon Biotech, China) and subcloned into the reconstituted mammalian expression vectors pcDNA3.1, named as pcDNA-antiDNA-LC and pcDNA-antiDNA-HC, respectively. The plasmids were co-transfected into CHO cells cultured in CHOGrow CD1 serum-free medium (SHANGHAI BASALMEDIA, China) by the Lipofectamine 3000 (Thermofisher, USA) according to manufacturer\u0026rsquo;s instructions, after 72 hours, the supernatants were harvested and for the next purification.\u003c/p\u003e \u003cp\u003eThe recombinant antibodies were purified using Protein G beads (Avantorscience, China), In brief, the Protein G resin (1ml) was equilibrated in PBS containing 0.1% NaN\u003csub\u003e3\u003c/sub\u003e, including equilibration, binding, washing steps. The elution in affinity chromatography was operated at the flow rate of 0.4 mL/min. Then 10ml of sample were loaded to the columns, followed by washing with 20 mL PBS containing 0.1% NaN\u003csub\u003e3\u003c/sub\u003e. Bound antibodies were eluted in 20 mL of 50 mM glycine-HCl (pH3.0), and immediately neutralized by addition of 10% volume of 1 M Tris\u0026ndash;HCl (pH8.0). Antibody concentrations were determined by BCA protein Assay Kit (Sangon Biotech, China), and the Purity of antibody were estimated by SDS-PAGE.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSite-Directed mutagenesis of the anti-DNA antibody\u003c/h2\u003e \u003cp\u003eSite-directed mutagenesis was performed to produce site-specific mutations at F33L or other positions in anti-DNA antibody. The pcDNA-antiDNA-LC or pcDNA-antiDNA-HC plasmid described above and the mutagenic primers shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (synthesized in Sangon Biotech) were used for this mutagenesis. In brief, PCR amplification was performed with the following 50 \u0026micro;L reaction mixture including 1 \u0026micro;L containing 50 ng of the plasmid was used as the template; 1 \u0026micro;L, containing 125 ng each, of both the forward and reverse primers; 1 \u0026micro;L dNTPs, 5 \u0026micro;L 10\u0026times;reaction buffer, 0.5 \u0026micro;L KOD polymerase (Toyobo, Japan) and 40 \u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. One cycle at 95\u0026deg;C for 2 min, followed by 16 cycles in which exposure 95\u0026deg;C for 30 s, 68\u0026deg;C for 8 min were alternated, was used for the PCR amplification. Amplification was followed by add 1uL DpnI (Thermofisher, USA) digestion by incubation at 37\u0026deg;C for 1 h to digest the template DNA. Next, after 10min heating at 80\u0026deg;C, 1 \u0026micro;L of the DpnI (Thermofisher, USA) digest reaction was transformed in to Top10 Competent Cells (Sangon biotech, China) using heat shock at 42\u0026deg;C for 45 s. Cells were then plated on LB agar plates containing 30 \u0026micro;g/mL kanamycin and incubated at 37\u0026deg;C overnight. A single transformed colony was grown in 10 mL of LB medium, containing Ampicillin (Sangon biotech, China) at a concentration of 50 \u0026micro;g/mL, in a shaking incubator at 200 rpm at 37\u0026deg;C overnight. The plasmid was isolated using the mini-prep plasmid isolation kit (Omega, China) and the mutation was confirmed by DNA sequencing in the commercial company (Sangon biotech, China).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimers used in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutation site\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimers sequences (5' to 3')\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eF33Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF:AGCATCAGCAGC\u003cb\u003eTAC\u003c/b\u003eTACTGGAGCTGGATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR:CCTGATCCAGCTCC\u003cb\u003eAGT\u003c/b\u003eAGTAGCTGCTGATGCT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eF33A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF:AGCATCAGCAGCTAC\u003cb\u003eGCC\u003c/b\u003eTGGAGCTGGATCAGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR:CCTGATCCAGCTCCA\u003cb\u003eGGC\u003c/b\u003eGTAGCTGCTGATGCT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eW98A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF:GCCAGGCACAGGAAC\u003cb\u003eGCC\u003c/b\u003eCTGTTCGACTACTGG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR:CCAGTAGTCGAACAG\u003cb\u003eGGC\u003c/b\u003eGTTCCTGTGCCTGGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eY95A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF:AGCTACGCCGGCAGC\u003cb\u003eGCC\u003c/b\u003eACCTACGTGTTCGGC\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR:GCCGAACACGTAGGT\u003cb\u003eGGC\u003c/b\u003eGCTGCCGGCGTAGCT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBinding ability assay\u003c/h2\u003e \u003cp\u003eIn brief, ELISAs were performed with Maxisorp plates (Thermofisher, USA) coated with 50 \u0026micro;l ssDNA(20-mer T)at a concentration of 10 \u0026micro;g/mL in PBS by overnight incubation. The plate was washed five times with PBS. 50 \u0026micro;l purified antibodies were transferred into the ELISA plate and incubated at 25\u0026deg;C for 2 h. Followed by five times washing with PBST, 100ul of HRP-conjugated anti-human IgG antibody diluted 1/5000 with 2% BSA-PBST was added to each well. The plate was incubated at 25\u0026deg;C for 1 h, followed by washing with PBST. The plate was incubated in the presence of 0.1 mL of 3,3\u0026prime;,5,5\u0026prime;-tetramethylbenzidine (TMB) substrate solution (Solarbio, China) at 25\u0026deg;C for 15 min. and the reaction was terminated by the addition of 0.1 mL of 0.3 M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Optical densities (OD) were measured at 405 nm using a microplate reader (SYNERGY H4, BioTeK, USA)\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe anti-ssDNA antibody has the innate conformation for binding to the ssDNA\u003c/h2\u003e \u003cp\u003eThe apo (non-binding form) and ssDNA binding forms of anti-ssDNA antibody has been identified by the X-ray crystallography. Their crystal structures, were downed from the protein database bank and with IDs as 5GKR and 5GKS, respectively [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], which were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B. The detailed binding behavior of antibody and ssDNA were analyzed and visualized, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, a considerable number of non-bonding interactions between the ssDNA and the antibody were found, e.g. the H-bonds were formed between the amino acid residues Y50, S54, S56, N58, H95 from Heavy chain and the ssDNA, and the Y95 and Y96 from the light chain could form the H-bonds with the ssDNA through their hydroxy groups. In addition, the pi-pi stacking interactions were observed between the aromatic group of side chains of residues and the base groups of the ssDNA, which are Y95\u003csub\u003eL\u003c/sub\u003e with the dT2 of the ssDNA, W98\u003csub\u003eH\u003c/sub\u003e, F33\u003csub\u003eL\u003c/sub\u003e with the dT3 of ssDNA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore whether the ssDNA binding affects the arrangement orientation of amino acid residues on the anti-DNA antibody, the key residues derived from the crystal structures of apo (5GKS) or ssDNA binding form (5GKR) were analyzed and the results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F. As shown, the residual orientation between two forms (apo vs ssDNA binding) was consistent and the corresponding RMSD was 0.481 \u0026Aring;, which suggests that the orientation of the key residues would not be influenced by the ssDNA binding. And moreover, the overlapping of CDR loops was also performed to explore the impact on the conformation change of CDR loops due to ssDNA binding, The obtained results showed that no obvious deviation was observed in the CDR loops between apo and ssDNA binding antibody forms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Especially for the H-CDR3, very few or no visible conformation changes were observed after ssDNA binding, take together, these observations suggested that the conformation of this anti-DNA antibody CDR have not been influenced by the binding to antigen ssDNA, implying that this anti-ssDNA antibody has the innate conformation for binding to the ssDNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCharacterize the influence of F33Y mutation on the conformation change of the Variable fragment region\u003c/h2\u003e \u003cp\u003eIt was reported that single-point mutation F33Y in the heavy chain of anti-ssDNA antibody could abolish its ssDNA binding ability [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To investigate the structural change of anti-ssDNA antibody caused by F33Y mutation, 200ns-molecular dynamic simulations procedures were employed. Firstly, the RMSD analysis was performed to evaluate the average amount of movement of backbone atoms throughout the entire protein structure, and the corresponding RMSD values fluctuation were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. As we can see, the RMSD value of the heavy chain of apo-anti-ssDNA antibody mainly varied between the 1.6\u0026Aring; and 2.8 \u0026Aring;, whereas, the light chain showed relatively high variation of the RMSD value mainly between the 1.8\u0026Aring; and 4\u0026Aring;. In contrast, the RMSD values of the Y33F mutant heavy chain and light chain showed similar variation (between the 1.6\u0026Aring; and 2.8\u0026Aring;). In addition, the superimposition analysis was performed between the crystal structure and the MD-optimized structure, as can be seen in the MD-optimized wildtype anti-ssDNA antibody has only small conformational change relative to the crystal structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, the overall architecture of F33Y mutant is similar to the crystal structure of wildtype antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These data revealed that F33Y single-point mutation had less or none significant influence on the architecture of this antibody.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess the conformational flexibility of the wildtype and mutant antibody, the root mean square fluctuations (RMSF) analysis were performed. The RMSF values were calculated by measuring atomic fluctuations after superimposing each structure of a MD trajectory onto the initial structure by means of least-squares fitting, to remove rotational and translational motion [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The results were shown that the corresponding RMSF value variation of amino acids located on the H-CDR1, H-CDR2 and H-CDR3, had the similar trend both in the wildtype and F33Y mutant antibodies, and that the value slightly decreased closed to the 72th residues of F33Y mutant heavy chain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), presumably because this region orientated outside of the antibody leading to more flexibility. Interestingly, no obvious difference in the RMSF value was observed in the 33th residues where the mutation F33 to Y happened. However, the RMSF value in the constant 1 region (CH1) of F33Y mutant heavy chain was smaller than that of wildtype, which indicates that the CH1 may be influenced by the H-F33Y mutation although this region is \u0026ldquo;spatially\u0026rdquo; far away from the mutation site. For the light chain of the antibody, similar trends were observed that the overall fluctuation of RMSF of the F33Y mutant were similar to those in the wildtype antibody, but some regions were exception, e.g, RMSF values of the resides in the F33Y mutant light chain CDR1 (L-CDR1) was less, whereas the values of L-CDR3 and L-CH1 is larger than those in wildtype. Taken together, these results demonstrated that the F33Y mutation in the heavy chain of anti-ssDNA antibody had more impact on the flexibility/mobility of light chain than heavy chain.\u003c/p\u003e \u003cp\u003ePCA helps to determine the most significant motion in dynamics trajectory. It was also carried out to investigate the important motions during the dynamic simulation process, and the covariance matrix of atomic fluctuations was diagonalized for predicting the eigenvalues. As the first few eigenvectors play a central role in the motions of protein, thus the first two eigenvectors of wildtype and F33Y mutant antibody were shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F, respectively, as we can see, there exist significant differences in the CDR loops of the light chain, in comparison, less difference existed in the H-CDR loops. But for the constant domains (C\u003csub\u003eH\u003c/sub\u003e1-C\u003csub\u003eL\u003c/sub\u003e), the motion of the top 2 eigenvectors are quite different between wildtype and F33Y mutant antibodies, it is suggested that F33Y mutation can influence the dynamic behaviors of the CH1 region of antibody.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe mutation F33Y impairs the ssDNA binding ability of anti-DNA antibody\u003c/h2\u003e \u003cp\u003eTo further explore the influence of the F33Y mutation on the ssDNA binding of this antibody, the MD simulation analysis was employed to imitate the dynamic process of wildtype and mutant antibody binding to the ssDNA. After 200ns MD simulation, the RMSD of the Cα for the wildtype antibody-ssDNA complex and F33Y mutant antibody-ssDNA complex were detected and collected. After binding to ssDNA, the RMSD values of heavy chains in wild-type antigen-antibody complex was mostly distributed between 2 \u0026Aring; and 3 \u0026Aring;, while the RMSD values of the light chains and the ssDNA in the complex were mostly between 1.6 \u0026Aring; and 2.4 \u0026Aring; and between 0.8 \u0026Aring; and 1.6 \u0026Aring;, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). which indicates that the conformation of ssDNA has slightly change in relevant to its initial crystal structure. In contrast, for the F33Y mutant antibody-ssDNA complex, although the RMSD values of the heavy chain were still between 2 \u0026Aring; to 3 \u0026Aring;, both the light chain and ssDNA had increased, especially it reached to 2.2 \u0026Aring; or above for the ssDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). which indicated that great conformation change happened in ssDNA during its binding to F33Y mutants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further evaluate the impact of F33Y mutation on antibody heavy and light chains, RMSF analysis were performed. The obtained results were shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the superimposition of RMSF plot curves between the F33Ymutant and WT antibodies fitted very well, means that no obvious difference were observed. But for the ssDNA, the RMSF plot of ssDNA in the mutant complex was lower than that in the wildtype complex, which indicated that ssDNA suffered more limitation in the mutant complex.\u003c/p\u003e \u003cp\u003eMotivated by these observations, the equilibrated snapshots of wildtype and mutant antibody-ssDNA complexes were collected and compared them with the corresponding initial structures of MD. The superimposition structure was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, obviously, there is no visual differences observed throughout the MD simulation for the WT and mutant complex. But PCA analysis on the dynamic behaviors of the mutant and wildtype antibody showed the CH1 region of the light chain shows quiet different orientation of movement between the WT and F33Y mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eFinally, the binding manner between the ssDNA and antibody were explored and shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, as we can see, when the F33 mutated to Y, the hydroxy group of Y33 can hydrogen-bond with the thymine base of ssDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), where F33 in the wildtype antibody cannot form the H-bond with the ssDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). and the dT2 formed more H-bonds in F33Y mutant than in wildtype. Hence, these observations clearly showed that the F33Y mutation could change binding manner between the antibody and ssDNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eF33Y mutation undermine/impair the interaction energy between antibody and ssDNA\u003c/h2\u003e \u003cp\u003eTo investigate the potential interaction mechanism between the ssDNA and antibody based on the energetic changes, the binding free energy of all ssDNA-antibody complexes was evaluated using the MM-GBSA methodology. The predicted binding free energies together with their corresponding energy contributions are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As we can see, the major contribution to stabilize the ssDNA-antibody complex is the electrostatic energy(ΔE\u003csub\u003eele\u003c/sub\u003e), However, comparing the ΔE\u003csub\u003eele\u003c/sub\u003e values between the wildtype (-78.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 kcal/mol) and F33Y mutant (-72.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5kcal/mol) antibodies, it showed that there was no significant difference between them, which suggests that F33Y mutation has weak influence on the electrostatic interactions between the antigen ssDNA and the antibody. The second contributor for the stabilization of ssDNA-antibody complex are van der Waals(vdW) forces (ΔE\u003csub\u003evdW\u003c/sub\u003e), which mainly form non-polar stacking interaction and play a crucial role in the binding between the protein and nucleotide[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The results showed that ΔE\u003csub\u003evdW\u003c/sub\u003e declined from \u0026minus;\u0026thinsp;54.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6 kcal/mol in wildtype antigen-antibody complex to -41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1kcal/mol in mutant complex (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which indicates that F33Y mutation in H-CDR1 resulted in significant decrease in vdW interaction. whereas for other energetic items (ΔE\u003csub\u003eGB\u003c/sub\u003e and ΔE\u003csub\u003eSA\u003c/sub\u003e), the difference is less significant in compared with ΔE\u003csub\u003evdW\u003c/sub\u003e. So, it can be concluded that the loss of binding ability of F33Y mutant antibody to ssDNA was largely due to the loss the vdW interaction between the antigen ssDNA and F33Y mutant antibody.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding free energies between antibody and ssDNA (kcal∙mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) obtained via the MM/GBSA approach for the two models.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntibody\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAntigen\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eEnergy components\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔE\u003csub\u003eele\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eΔE\u003csub\u003evdW\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eΔE\u003csub\u003eGB\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eΔE\u003csub\u003eSA\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eΔE\u003csub\u003etot\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eWT\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003essDNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-78.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-54.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e80.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-6.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e-58.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF33Y\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003essDNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e-72.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e-41.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e74.2\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e-5.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c7\"\u003e \u003cp\u003e-44.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eΔE\u003csub\u003etot\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;ΔE\u003csub\u003eele\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔE\u003csub\u003evdW\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔE\u003csub\u003eGB\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;ΔE\u003csub\u003eSA\u003c/sub\u003e. The stand errors of the mean are listed in parentheses.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the energy contributions of individual residues, the residue-based free energy decomposition analysis was performed to calculate the energy of residues in the CDR of antibody heavy and light chain. The results showed that the interactions of 23 residues Y32, F33, Y50, I51, Y52, Y53, S54, G55, S56, T57, N58, Y59, K64, R94, H95, R96, N97, W98 in the heavy chain, and Y32, S94, Y95, Y96 in the light chain, were identified as contributor to the binding with the ssDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For the wildtype, the residues F33\u003csub\u003eH\u003c/sub\u003e, W98 \u003csub\u003eH\u003c/sub\u003e, and Y96\u003csub\u003eL\u003c/sub\u003e were identified as the top three contributors for the ssDNA binding of the antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For the F33Y mutant antibody-ssDNA complex, the residues Y/F33 \u003csub\u003eH\u003c/sub\u003e, W98 \u003csub\u003eH\u003c/sub\u003e, and Y95\u003csub\u003eL\u003c/sub\u003e were identified as key contributors. These 23 residues could be divided into four groups according to their properties, 11 amino acids in aromatic groups (11/23), 6 in polar groups (6/23), 3 in charged groups (3/23) and 3 in nonpolar group (3/23) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Comparing the energy changes among groups, the aromatic groups contribute the most, followed by polar group and charged group, which further demonstrated that the electrostatic interactions does not play a critical role during the antibody-antigen binding process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe energy contribution of residues of antibody to its binding with the ssDNA\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eThe properties of amino acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003eEnergy contribution (kcal/mol)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eF33Y\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"10\" rowspan=\"11\"\u003e \u003cp\u003e\u003cb\u003eAromatic amino acids\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"10\" rowspan=\"11\"\u003e \u003cp\u003e-18.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-4.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"10\" rowspan=\"11\"\u003e \u003cp\u003e-14.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-CDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-4.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF/Y33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-3.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-CDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eH95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-CDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-FRW3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e\u003cb\u003ePolar amino acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e-8.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e-5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-FRW3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-FRW3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-1.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-1.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL-CDR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eCharged amino acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-0.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-FRW3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eK64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eR94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eNonpolar amino acid\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eG55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eH-CDR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eL99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eFurthermore, analyzing the location of these residues found that they were mainly located in the heavy chain CDR1 (H-CDR1, 2/23), CDR2 (H-CDR2, 7/23), CDR3 (H-CDR3, 6/23), framework 3 (H-FRW3, 4/23), and light chain CDRs (L-CDRs, 4/23) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For the wildtype, the H-CDR2 contributed greatest in compared to other regions, followed by the H-CDR3, then L-CDRs, H-CDR1 and H-FRW3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). For the F33Y mutant, H-CDR2 and H-CDR3 have the greatest contributions to the ssDNA binding, followed by H-CDR1 and L-CDRs, then H-FWR3. Yet it is worth noting that the binding energies of H-FR3, H-CDR2, and L-CDRs were largely reduced in F33Y mutant complex comparing with wildtype. Taken together, these results implied that the residues located in these regions might be the most crucial for the binding of antibodies to ssDNA, and that F33Y mutation causing the antibody to lose its antigen binding ability was achieved by changing the energy of the key amino acids in these regions.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePi-pi stacking interaction but not hydrogen bonds between antibody and ssDNA was undermined/impaired upon F33Y mutation\u003c/b\u003e \u003c/p\u003e \u003cp\u003ePrevious study showed that the aromatic pi-pi stacking interaction, a major type of vdW force, was one of the most fundamental interaction forces for the high affinity and specificity of autoantibody [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We identified Y/F33\u003csub\u003eH\u003c/sub\u003e, W98\u003csub\u003eH\u003c/sub\u003e and Y95\u003csub\u003eL\u003c/sub\u003eas key amino acid residues for ssDNA binding in both wild-type and mutant antibody. All of them have aromatic cyclic structures, indicating that they are likely to form stable pi-pi interactions with DNA bases. To verify this, the non-bond interactions detection analyses were performed to inspect the pi-pi stacking interactions between the key residues and the nucleobase of ssDNA. The results showed that F33\u003csub\u003eH\u003c/sub\u003e, W98\u003csub\u003eH\u003c/sub\u003e and Y95\u003csub\u003eL\u003c/sub\u003e could form the stable pi-pi stacking interaction with the nucleotide base group of ssDNA in the wildtype antibody during the whole MD process (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). While for the F33Y mutant, the Y33\u003csub\u003eH\u003c/sub\u003e and W98\u003csub\u003eH\u003c/sub\u003e could also form the stable pi-pi interaction with the ssDNA, which were similar in the wildtype. However, for the Y95\u003csub\u003eL\u003c/sub\u003e in the F33Y mutant, the distance between dT2 and Y95 (\u003cem\u003ed\u003c/em\u003e\u003csub\u003edT2\u0026minus;\u0026minus;\u0026minus;Y95\u003c/sub\u003e) suddenly increased from the 4 \u0026Aring; to more than 5 \u0026Aring; (critical distance of pi-pi stacking is less than 5 \u0026Aring;) at the end of the MD process, which means that this pi-pi stacking was finally impaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Moreover, it also could see that the dT2 ring of the ssDNA was sandwiched by the F33 and W98 to form the parallel pi-pi-pi stacking interaction in the wildtype antibody-ssDNA complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). However, F33Y mutant, the parallel sandwich pi-pi-pi stacking were destroyed due to that the Y33 cannot form the pi-pi stacking interaction with the dT2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Hence, these results demonstrated that F33Y mutation weaken the pi-pi stacking interactions between the antibody and ssDNA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHydrogen bond interaction is also one of the most important non-bonding interactions between the antibody and antigen. To explore whether the hydrogen bond interaction contributes to the ssDNA binding of the wild type and the mutant antibody, the MD simulation timescale was performed. The results showed that Y50\u003csub\u003eH\u003c/sub\u003e, T57\u003csub\u003eH\u003c/sub\u003e, Y96\u003csub\u003eH\u003c/sub\u003e, and W98\u003csub\u003eH\u003c/sub\u003e could form the same number of stable hydrogen bonds with ssDNA in both the wild type and the mutant (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that the mutation did not alter the way and quantity of these residues binding to DNA. S56\u003csub\u003eH\u003c/sub\u003e could form more hydrogen bonds in the wild type, but the N58\u003csub\u003eH\u003c/sub\u003e residue could form more stable hydrogen bonds with ssDNA in the F33Y mutant. Additionally, although Y33\u003csub\u003eH\u003c/sub\u003e and S54\u003csub\u003eH\u003c/sub\u003e in the mutant could form additional hydrogen bonds with ssDNA, these hydrogen bonds had very short duration throughout the entire MD process, implying that these bonds may not be the contributors to the overall stability of the binding process.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIntermolecular hydrogen bonds between ssDNA and antibody\u003csup\u003ea\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eWT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c11\" namest=\"c7\"\u003e \u003cp\u003eF33Y\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eAcceptor\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003eDonor\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOcpy\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDist\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAng\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eAcceptor\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eDonor\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eOcpy\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eDist\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAng\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eY50\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT3@O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY50\u003csub\u003eH\u003c/sub\u003e@HH Y50\u003csub\u003eH\u003c/sub\u003e@OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e164.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT3@O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eY50\u003csub\u003eH\u003c/sub\u003e@HH Y50\u003csub\u003eH\u003c/sub\u003e@OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e99.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e163.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eY96\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT3@O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eY96\u003csub\u003eL\u003c/sub\u003e@HH Y96\u003csub\u003eL\u003c/sub\u003e@OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e160.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT3@O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eY96\u003csub\u003eL\u003c/sub\u003e@HH Y96\u003csub\u003eL\u003c/sub\u003e@OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e98.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e160.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eW98\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT3@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eW98\u003csub\u003eH\u003c/sub\u003e@H W98\u003csub\u003eH\u003c/sub\u003e@N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e154.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT3@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eW98\u003csub\u003eH\u003c/sub\u003e@H W98\u003csub\u003eH\u003c/sub\u003e@N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e95.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e154.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eT57\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT1@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT57\u003csub\u003eH\u003c/sub\u003e@H T57\u003csub\u003eH\u003c/sub\u003e@N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e147.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT1@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eT57\u003csub\u003eH\u003c/sub\u003e@H T57\u003csub\u003eH\u003c/sub\u003e@N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e53.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e142.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eT57\u003csub\u003eH\u003c/sub\u003e@O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edT2@H3 dT2@N3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e151.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eT57H@O\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edT1@H3 dT1@N3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e54.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e146.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eS56\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT2@O4'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS56\u003csub\u003eH\u003c/sub\u003e@HG S56\u003csub\u003eH\u003c/sub\u003e@OG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e146.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT2@O4'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS56\u003csub\u003eH\u003c/sub\u003e@HG S56\u003csub\u003eH\u003c/sub\u003e@OG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e56.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e148.47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT1@O3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eS56\u003csub\u003eH\u003c/sub\u003e@HG S56\u003csub\u003eH\u003c/sub\u003e@OG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e144.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eN58\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003edT2@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN58\u003csub\u003eH\u003c/sub\u003e@HD21 N58\u003csub\u003eH\u003c/sub\u003e@ND2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e162.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT2@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN58\u003csub\u003eH\u003c/sub\u003e@HD21 N58\u003csub\u003eH\u003c/sub\u003e@ND2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e99.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e162.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN58\u003csub\u003eH\u003c/sub\u003e@OD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003edT2@H3 dT2@N3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e162.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN58\u003csub\u003eH\u003c/sub\u003e@OD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edT2@H3 dT2@N3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e96.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e161.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN58\u003csub\u003eH\u003c/sub\u003e@OD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003edT1@H3 dT1@N3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e28.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e136.85\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eY33\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT2@O3'\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eY33\u003csub\u003eH\u003c/sub\u003e@ HH Y33\u003csub\u003eH\u003c/sub\u003e@ OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e36.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e138.13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT2@O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eY33\u003csub\u003eH\u003c/sub\u003e@ HH Y33\u003csub\u003eH\u003c/sub\u003e@ OH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e35.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e146.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eS54\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003edT2@OP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS54\u003csub\u003eH\u003c/sub\u003e@HG S54\u003csub\u003eH\u003c/sub\u003e@OG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e45.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e163.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003ea\u003c/sup\u003e The subscripted H and L indicate the residues reside in the antibody heavy and light chains, respectively.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003eb\u003c/sup\u003e Hydrogen bond occupancy during MD (%).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003ec\u003c/sup\u003e Time averaged hydrogen bond length (\u0026Aring;).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"11\"\u003e\u003csup\u003ed\u003c/sup\u003e Time averaged hydrogen bond angle (\u0026ordm;).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePi-pi stacking interactions dominated the binding between antibody and ssDNA\u003c/h2\u003e \u003cp\u003eIn order to confirm the dominated role of the pi-pi stacking interaction between the anti-DNA antibody and the ssDNA, the F33Y\u003csub\u003eH\u003c/sub\u003e, F33A\u003csub\u003eH\u003c/sub\u003e, W98A\u003csub\u003eH\u003c/sub\u003e, Y95A\u003csub\u003eL\u003c/sub\u003e mutants and the wildtype antibody were expressed and purified (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Their binding performance towards the ssDNA were evaluated by the ELISA experiments. The corresponding results showed that almost all the mutants lost the ssDNA binding ability (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) due to impairing of pi-pi stacking interaction, which further demonstrated that pi-pi stacking was one of the key contributors for this anti-ssDNA antibody.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDuring the recognition and binding process of antibodies toward antigen, each portion of the antibody variable region plays different effects in the identification and binding antigen molecules, especially the CDR3 on the heavy chain, which is recombinantly recombined by the VDJ encoding an antibody gene. It is believed that the H-CDR3 of the antibody plays a decisive role in identifying antigen and binding antigen. So in this study, MD simulation results showed that there are less/none influence on the contribution of H-CDR3 to binding with ssDNA upon introducing mutation F33Y in the H-CDR1, And together with the analysis on the crystal structures of the complex (antibody-ssDNA complex) and apo-antibody which showed that no obvious difference in the conformation of the CDRs were observed upon on the binding with ssDNA, indicating that the antibody may have a tendency to be born with binding to ssDNA.\u003c/p\u003e \u003cp\u003eAccording to our finding, the contribution of different regions on binding to ssDNA is in this order: H-CDR2\u0026thinsp;\u0026gt;\u0026thinsp;H-CDR3\u0026thinsp;\u0026gt;\u0026thinsp;L-CDRs\u0026thinsp;\u0026gt;\u0026thinsp;or =\u0026thinsp;H-CDR1\u0026thinsp;\u0026gt;\u0026thinsp;H-FRW3, that means that H-CDR2 also play similar important role in binding with the antigen ssDNA as compared with the H-CDR3. H-FRW3 was traditionally considered as the scaffold to support the CDRs, but it is found to interact with antigen directly in this study. This may be the particular characteristics of this anti-ssDNA antibody, and anti-DNA antibodies may take other ways to achieve identification and binding of ssDNA. When the H-F33 mutate to Y, the contribution from the HCDR2 and H-FRW3 of F33Y mutant were significantly decreased to almost half of that in the wildtype. But the other regions were not largely influenced by this mutation. These results can at least partly explain that why F33Y mutant lost the binding ability toward the antigen ssDNA.\u003c/p\u003e \u003cp\u003eAdditionally, the function of H-FRW3 was need to be recognized since it was found to interact with antigen directly, which was traditionally considered just as the scaffold to support the CDRs. This results is consisted with the Victor Ovchinnikov\u0026rsquo;s findings, that high frequency mutation in the H-FRW3 can expand diversity of anti-HIV neutralizing antibody [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], implied that the H-FRW3 not only act as the scaffold to support the CDRs, but also affect the binding properties of CDR toward the antigen. And in this study, the mutation (F33Y) located in the H-CDR1, which is spatially separated from the H-FRW3, but this mutation significant influence the binding of H-FRW3 to the antigen ssDNA possible through the allosteric effect.\u003c/p\u003e \u003cp\u003ePrevious studies pointed out the CH1 domain influences the antibody paratope and in turn its specificity and functional activities [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and, Lavoie et.al found that the Fv\u0026ndash;HEL complex was found to have a dissociation constant one order of magnitude lower than that of the Fab\u0026ndash;HEL complex [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, CH1 loops implicated in interactions between H and L chains which show the most consistent and substantial changes upon Ag binding [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] in this study, we found that the mutation (F33Y) located in the H-CDR1 can influence the dynamic behavior of the CH1 region of the Fab. PCA analysis on the MD trajector clearly showed that the movement of CH1 showed quiet different behavior upon inducing F33Y mutation, which further verified that the C and V regions are structurally coupled and affect each other mainly by the Allosteric effects, So this study can give us hint in antibody humanized engineering, just graft the CDRs may lead to lost its original ability of antibody.\u003c/p\u003e \u003cp\u003eIn previous years, Tanner and co-workers identified a conserved and fundamental structural element responsible for the recognition of ssDNA, termed the \u0026ldquo;ssDNA-antibody recognition module\u0026rdquo; (D-ARM) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The D-ARM consists of a tyrosine residue that stacks with the base and a glycine that forms a hydrogen bond with the base. Thus, the H-bond interaction and aromatic group stacking interaction are the main contributor for the autoantibody binding to its antigen. In present study, it was found that F33Y mutation in H-CDR1 can eliminate the ssDNA binding ability of this antibody, the H-bonding interaction between the mutant antibody and ssDNA were not weaken in compared with the wildtype antibody, but the pi-pi stacking interaction derived from the aromatic residues and base groups of DNA were significantly impaired upon the F33Y mutation, also the experimental results showed that any one mutation in three key residues (F33\u003csub\u003eH\u003c/sub\u003e, W98\u003csub\u003eH\u003c/sub\u003e, and Y95\u003csub\u003eL\u003c/sub\u003e) lead to the loss of ssDNA binding ability of mutant antibody. these findings at least partly indicated that pi-pi stacking interaction between the antibody and antigen is one of major factor that determine the whether the antibody can bind with DNA. These results are consistent with previous study that the three Aromatic Residues W56, W75 and F79 form a stable pi-pi stacking effect for ssDNA-binding Protein [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the Jiangsu University, China (18JDG028).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.N. \u0026nbsp;and D.C. analyzed the data, prepared figures 1- 6 \u0026nbsp; and wrote the main manuscript. Y.X., \u0026nbsp; H.Z. and F. S. collected the data and prepared figures 7 and tables 1-4. \u0026nbsp;Z.W. contributed to scientific discussion and critical review of the manuscript. Z.N. \u0026nbsp;and D.C. conceived the idea and designed the structures of the paper. All authors contributed to the article and approved the submitted version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcInnes IB, Schett G (2011) The Pathogenesis of Rheumatoid Arthritis. New Engl J Med 365: 2205-2219. doi:/ 10.1056/NEJMra1004965\u003c/li\u003e\n\u003cli\u003evan Regenmortel MH (2000) The recognition of proteins and peptides by antibodies. J Immunoassay 21: 85-108. doi:/10.1080/01971520009349530\u003c/li\u003e\n\u003cli\u003eLeal E, Jaloma-Cruz ARBarros-Nunez P (2004) High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemia. 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(2018) Role of framework mutations and antibody flexibility in the evolution of broadly neutralizing antibodies. Elife 7. doi:/10.7554/eLife.33038\u003c/li\u003e\n\u003cli\u003eLavoie TB, Drohan WNSmith-Gill SJ (1992) Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme. J Immunol 148: 503-13\u003c/li\u003e\n\u003cli\u003eSela-Culang I, Kunik VOfran Y (2013) The structural basis of antibody-antigen recognition. Front Immunol 4: 302. doi:/10.3389/fimmu.2013.00302\u003c/li\u003e\n\u003cli\u003eTanner JJ, Komissarov AADeutscher SL (2001) Crystal structure of an antigen-binding fragment bound to single-stranded DNA. J Mol Biol 314: 807-22. doi:/10.1006/jmbi.2001.5178\u003c/li\u003e\n\u003cli\u003eGamsjaeger R, Kariawasam R, Gimenez AX, et al. (2015) The structural basis of DNA binding by the single-stranded DNA-binding protein from Sulfolobus solfataricus. Biochem J 465: 337-46. doi:/10.1042/BJ20141140\u003c/li\u003e\n\u003cli\u003eKhamassi M, Xu L, Rey J, et al. (2020) The CH1alpha domain of mucosal gp41 IgA contributes to antibody specificity and antiviral functions in HIV-1 highly exposed Sero-Negative individuals. PLoS Pathog 16: e1009103. doi:/10.1371/journal.ppat.1009103\u003c/li\u003e\n\u003cli\u003eLi J, Wang Y, An L, et al. (2018) Direct Observation of CH/CH van der Waals Interactions in Proteins by NMR. J Am Chem Soc 140: 3194-3197. doi:/10.1021/jacs.7b13345\u003c/li\u003e\n\u003cli\u003eVictora GDNussenzweig MC (2022) Germinal Centers. Annu Rev Immunol 40: 413-442. doi:/10.1146/annurev-immunol-120419-022408\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"autoantibody, molecular dynamic simulation, mutation, pi-pi stacking, repertoire","lastPublishedDoi":"10.21203/rs.3.rs-4446391/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4446391/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA recent study showed that just one point mutation F33 to Y in the H-CDR1 could lead to the autoantibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen ssDNA, and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33\u003csub\u003eH\u003c/sub\u003e, W98\u003csub\u003eH\u003c/sub\u003e and Y95\u003csub\u003eL\u003c/sub\u003e in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA, which could be verified by the result that any functional mutation in three key residues (F33\u003csub\u003eH\u003c/sub\u003e, W98\u003csub\u003eH\u003c/sub\u003e, and Y95\u003csub\u003eL\u003c/sub\u003e) could lead to the loss of ssDNA binding ability of mutant antibody. Our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.\u003c/p\u003e","manuscriptTitle":"Mechanistic insights into how the single point mutation change the autoantibody repertoire","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-03 19:49:21","doi":"10.21203/rs.3.rs-4446391/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-30T14:28:43+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-30T12:28:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-28T21:12:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117436602345975563937877815018570412507","date":"2024-05-22T17:05:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192295208106855319545370984886236360128","date":"2024-05-22T16:03:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-22T14:21:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T07:25:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T07:25:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Protein Journal","date":"2024-05-20T03:28:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-protein-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jopc","sideBox":"Learn more about [The Protein Journal](http://link.springer.com/journal/10930)","snPcode":"10930","submissionUrl":"https://submission.nature.com/new-submission/10930/3","title":"The Protein Journal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"dab078ab-83db-45b2-8135-7c51f08361df","owner":[],"postedDate":"June 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-07-30T04:02:22+00:00","versionOfRecord":{"articleIdentity":"rs-4446391","link":"https://doi.org/10.1007/s10930-024-10225-w","journal":{"identity":"the-protein-journal","isVorOnly":false,"title":"The Protein Journal"},"publishedOn":"2024-07-28 04:02:22","publishedOnDateReadable":"July 28th, 2024"},"versionCreatedAt":"2024-06-03 19:49:21","video":"","vorDoi":"10.1007/s10930-024-10225-w","vorDoiUrl":"https://doi.org/10.1007/s10930-024-10225-w","workflowStages":[]},"version":"v1","identity":"rs-4446391","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4446391","identity":"rs-4446391","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}