Structural Impacts of Two Disease-linked ADAR1 Mutants: A Molecular Dynamics Study

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Dysregulation of ADAR1 editing activity, often arising from genetic mutations, has been linked to elevated interferon levels and the onset of autoinflammatory diseases. However, understanding the molecular underpinnings of this dysregulation is impeded by the lack of an experimentally determined structure for the ADAR1 deaminase domain. In this computational study, we utilized homology modeling and the AlphaFold2 to construct structural models of the ADAR1 deaminase domain in wild-type and two pathogenic variants, R892H and Y1112F, to decipher the structural impact on the reduced deaminase activity. Our findings illuminate the critical role of structural complementarity between the ADAR1 deaminase domain and dsRNA in enzyme-substrate recognition. That is, the relative position of E1008 and K1120 must be maintained so that they can insert into the minor and major grooves of the substrate dsRNA, respectively, facilitating the flipping-out of adenosine to be accommodated within a cavity surrounding E912. Both the orthosteric R892 mutations of R892 and the allosteric Y1112F mutation alter K1120 position and ultimately hinder substrate RNA binding. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Adenosine deaminase acting on RNA (ADAR) is a class of enzymes to process the adenosine-to-inosine (A-to-I) RNA editing through deamination reaction, making it the most prevalent type of post-transcriptional nucleotide modification in humans [ 1 , 2 ]. Since inosine can form Watson-Crick base pairing with cytidine, the resulted inosine behaves like guanosine and, as a result, the A-to-I editing on RNA significantly modifies RNA function, such as miRNA biosynthesis and target recognition, splicing patterns, gene regulation, and the long-non-coding RNA functions [ 2 – 4 ]. There are three ADARs in humans, namely ADAR1, ADAR2, and ADAR3 [ 5 ]. Both ADAR1 and ADAR2 are catalytically active and expressed in almost all tissues, whereas ADAR3 is only expressed in brain and has no detectable catalytic activity. It has been reported that ADAR3 can compete with ADAR2 to inhibit RNA editing by directing its binding to GRIA2 pre-mRNA in glioblastoma [ 6 ]. Because ADAR1’s editing functions on dsRNA inhibits innate immunity and the interferon-mediated response, dysregulation of ADAR1 activity causes upregulated interferon and autoinflammatory diseases such as Aicardi-Goutieres syndrome (AGS) and dyschromatosis symmetrica hereditarian (DSH) [ 7 – 9 ]. AGS is a rare childhood autoimmune disorder that primarily affects the brain, immune system, and skin. [ 10 ] Currently it has been reported that seven genes, including ADAR1, confirmed to be associated with AGS [ 11 ]. The severity of the disease caused by the loss-of-function ADAR mutations may depend on the overall impact of these mutations on A-to-I RNA editing activity [ 12 , 13 ]. A decrease in editing activity below a certain threshold likely triggers the development of a neurological disorder [ 14 ]. Molecular dynamics (MD) simulations have long been a useful tool in providing structural and dynamic details of proteins [ 15 – 19 ]. In this work, we carried out MD simulations to study two AGS-causing mutations in ADAR1 deaminase domain, R892H and Y1112F, identifying their structural deviation from the wild-type (WT). From the N-terminus to the C-terminus, ADAR1 includes two Z-DNA binding domains (ZBDs), three dsRNA binding domains, and deaminase domain. Currently, the solved structure of ADAR1 is limited to the ZBDs [ 20 ]. We used homology modeling to build one apo ADAR1 deaminase domain, employing the solved human ADAR2 deaminase domain structure as a template. To enhance our conformational sampling, we also generated another ADAR1 structure using AlphaFold2, a method known for its high precision in protein structure prediction [ 21 ]. For each of the two model structures, the R892 residue was replaced by histadine to form two R892H strucures, and the Y1112 residue was replaced by phenealanine to form two Y1112F strucures. All of the six structures underwent MD simulations to refine and collect conformations, ultimately helping us deduce the structural criteria for RNA substrate recognition. Additionally, we retrieved a wild-type conformation from the AlphaFold2 trajectory, superimposed it with the RNA-bound ADAR2 structure, replaced the RNA sequence by HER1 RNA [ 22 ], and submitted it for MD simulations. Sequence alignment of ADAR1 and ADAR2 deaminase domains (Figure S1 ) indicates 39% sequence identity and 59% sequence similarity, suggesting that the available ADAR2 structural data provides useful guidance in envisioning how ADAR1 associates with a dsRNA substrate. The ADAR2 deaminase domain is composed of 20 loops, four of which come into contact with the dsRNA. As indicated in Fig. 1 a and Figure S1 , these loops are referred to as the base-flipping loop (E485’-G489’), the 5'-binding loop (A454’-R477’), the 3'-binding loop I (A347’-L352’), and the 3’-binding loop II (L584’-N597’) [ 23 ]. For ease of reading, ARAD2 residues are marked with primes to distinguish them from the unprimed ADAR1 residues. As shown in Fig. 1 b, ADAR2 catalytic site is enclosed by E396’, E488’, and K594. The E396’ (on the N-terminal end of α2 helix in the core structure) secures the flipping-out adenine (on the substrate RNA strand) base while the E488’ (on the base-flipping loop) inserts into the minor groove, filling the space left by the flipping-out adenine, and contacts the unpaired, orphaned base located on the complementary RNA strand. Taking the substrate RNA strand as a viewpoint, from top to bottom in Fig. 1 a, the 3’ end and 5’ end regions relative to the flipping-out base, which is to be edited, are in contact with the 3’-binding loops and 5’-binding loop, respectively. Because of its high flexibility, the 24-base-long 5’-binding loop structure was not resolved in the apo ADAR2 structure (PDB: 1ZY7) but was resolved in the RNA-ADAR2 complex (PDB: 5HP2) [ 24 , 25 ]. The R348’ (in the 3’-binding loop I ) and K594’ (in the 3’-binding loop II ) both stabilize the dsRNA. Except R348’ not conserved by ADAR1 G865, the catalytic site’s E396’, E488’, and K594’ are conserved by ADAR1’s E912, E1008, and K1120. The E912-E1008 and E1008-K1120 separation distances were adopted to categorize the collected WT, R892H, and Y1112F conformations. We hypothesized that if the E1008-K1120 distance exceeds the tolerance level, it would discourage the dsRNA recognition process, ultimately reducing the A-to-I editing activity. Numerous AGS-causing mutations are on the ADAR1 deaminase domain, including A870T (A353’ in ADAR2), I872T (V355’), R892H (K376’), K999N (Q479’), G1007R (G487’), Y1112F (A587’), and D1113H (E588’), as summarized in Figure S2. By analyzing these corresponding residues on the ADAR2 deaminase domain structure, we foresee the possible consequence of ADAR1’s mutations. For example, A353’ and V355’ are in vicinity of E396’ in ADAR2 and accordingly either A870T or I872T mutation impairs the interaction between E912 and the flipping base in ADAR1. Because G487’ is next to E488’ and K376’ holds salt bridges to secure the 3’-binding loop II , either G1007R or R896H mutation surely deforms the active site. Q479’ is in the C-terminus of the 5’-binding loop and thus the correlated K999N mutation may interfere the induced-fit process when associating the dsRNA substrate. A587’, E588’, and K594’ are located in the 3’-binding loop II ; thus Y1112F and D1113H mutations may pull K1120 away from the active site. Since ADAR1’s Y1112 and ADAR2’s A587’ possess dissimilar sizes and hydrophilicities, it is necessary to learn the role played by Y1112 before investigating Y1112F mutation. Our objective is to explore the susceptibility of ADAR1's active site. To achieve this, we selected an orthosteric site R892H mutation and an allosteric site Y1112F mutation. Our conformational analysis suggests that in R892H variant, the substituted H892 is not able to maintain the R892-E1123 ionic linkage. On the other hand, the hydrophobic F1112 in Y1112F mutant reduces its solvent-accessible surface area (SASA) and causes a contraction movement of the 3’-binding loop II toward the α1 helix. Both mutations rearrange K1120 position and interfere the substrate RNA recognition. 2. Methods 2.1 ADAR1 Structure Preparation We used the AMBER 18 software package for MD simulations. To build the initial WT structure, we employed the homology modeling module in Discovery Studio 2020 program [ 31 ], using the ADAR2 deaminase domain structure (PDB: 1ZY7) as a template [ 24 ]. This choice was supported by their 39% sequence identity and 59% sequence similarity. (Figure S1 ). It's important to note that the success of homology modeling in predicting protein structure greatly depends on sequence identity, with a typical criterion being around 30%. In addition, we assessed the model's accuracy and evaluated the correctness of the model prediction using the PROCHECK scoring algorithm, which enables a more precise evaluation of the reliability of the modeling results. To intensify our conformational sampling, a second prediction was carried out with AlphaFold2 method without assigning any template. We further compared the two predicted ADAR1 structures with the crystallography-solved apo ADAR2, excluding the 5'-binding loop, which was not resolved in the apo ADAR2 structure. This loop was observed in the ADAR2-RNA complex, suggesting an induced-fit conformational change upon substrate binding in ADAR2. The root-mean-square deviation (RMSD) calculated between the homology-built ADAR1 and the solved ADAR2 is 1.963, between the AlphaFold2-built ADAR1 and the solved ADAR2 is 4.446, and between the homology-built ADAR1 and AlphaFold2 ADAR1 is 4.691. Superpositions among the three structures (see Figures S3a-c) indicate that the deviations are primarily in the loops flanking the structure core. Since the flexible 5’-binding loop is expected to remain freely exposed to the solvent, we replaced all 24 amino acid residues with 24 sequential alanine residues and surrounded them with water molecules. This modification was made to keep the loop away from the core structure and minimize any structural biases. 2.2 ADAR1-RNA Complex Structure Preparation To investigate the engagement between our ADAR1 model and a dsRNA substrate in the catalytic site, we constructed an RNA-bound ADAR1 model by taking a frame from the AlphaFold2 trajectory at 1.6 µs due to its remarkable conformational similarity to the RNA-bound form of the ADAR2 deaminase. We superimposed it to the RNA-ADAR2 complex structure (PDB: 5HP2) and substituted the bound RNA sequence with a short segment of the HER1 RNA sequence [ 22 ]. Initially we constrained the RNA structure while allowing the ADAR1 structure to adapt to accommodate the substrate, and later we relaxed the entire complex to sample conformations. Since the simulation duration was short, the induced-fit process of the 5’-binding loop was not considered. 2.3 Molecular Dynamics Simulations Each studied structure was immersed in a cubic TIP3P water box, with the walls maintained at least 10 Å away from the immersed ADAR1 structure. The energy minimization of each solvated system was conducted in three stages, as in our previous studies [ 32 – 34 ]. The Particle-Mesh Ewald method was applied to handle long-range electrostatic interactions, and the SHAKE algorithm was used to constrain all bonds containing hydrogen atoms to their equilibrium lengths. An 8 Å cutoff distance was set for efficient simulation of nonbonding interactions such as short-range electrostatics and van der Waals interactions. Each simulated ADAR1 WT structure underwent a gradual heating process from 0 to 300 K over 100 ps, followed by a density equilibration procedure at the target temperature for 100 ps, and then constant equilibration at the target temperature for 1000 ps. Subsequently, the systems were subjected to a 2 µs simulation run. In the case of the two mutants, we selected a WT conformation at the 1 µs time frame and replaced R892 with histidine and Y1112 with phenylalanine to create the initial structures of the R892H and Y1112F variants, respectively. To maintain the electroneutrality of the studied systems, we added seven chloride ions to the WT and Y1112F simulations, six chloride ions to the R892H simulation, and 27 sodium ions to the ADAR1-RNA complex simulation. The setup and protocols mentioned above for WT’s heating, density equilibration, and equilibrium were also applied to process the four mutant trajectories and one RNA-bound complex trajectory. In the following these six trajectories are termed WT HM , WT AF , R892H HM , R892H AF , Y1112F HM , and Y1112F AF , based on the conducted homology modeling (HM) and AlphaFold2 (AF) approach. The length for each of the six apo ADAR1 trajectories is 2 µs and for the RNA-bound ADAR1 is 1 µs. 3. Result and Discussion 3.1 MD Stability Each of the WT, R892H variant, and Y1112F variant was simulated with two independent replicas for conformation collection. As indicated in Figure S4, RMSF (root mean square fluctuation) values plotted against amino acid residues show high flexibility in the 5’ end binding loop (residues 972–995), which was replaced by 24 alanine residues in each of the six trajectories. Consequently, the coordinates of the 5’ end binding loop were excluded from the RMSD (root mean square deviation) calculation. Our RMSD calculation was based on the heavy atoms throughout the entire 2 µs production duration. All the modeling systems show a rapidly increasing RMSD values and a deviation from their initial structure of approximately 3 Å (see Figure S5). As indicated in Figure S5a the WT HM trajectory reached 3.0 Å around 800 ns and maintained a stable centering at 3.2 Å between 800 and 2000 ns. Likely, in Figure S5b, the second half of the WT AF trajectory is stable, centering at 3.5 Å. The two R892H HM and R892H AF trajectories exhibit higher variation, reaching nearly 4 Å after 1100 ns from their initial structures (see Figure S5c and S5d). The Y1112F HM trajectory became stable around 1000 ns (see Figure S5e), whereas the Y1112F AF curve maintained a mean value of 3.4 Å between 900 and 1300 ns and increased to a mean value of 3.8 Å between 1300 and 2000 ns (see Figure S5f), implying a regional structural change along the trajectory. Following this, we captured snapshots from each trajectory, ranging from 1 µs to 2 µs, for structural investigation. 3.2 Conformational Discrepancy among the Variants Measured between the Cδ atom in the glutamate residue and the Cζ atom in the lysine residue, the E396’-E488’ separation distance is 14.6 Å in the apo ADAR2 structure and is 18 Å in the RNA-bound ADAR2 structure; the E488’-K594’ separation distance is 9.4 Å in the apo structure and is 15.1 Å in the RNA-bound structure. The condensed apo ADAR2 conformation could be attributed to the salt condition during crystallization or its intrinsic nature. Apparently, the apo ADAR2 conformation undergoes induced-fit relaxation when accommodating the dsRNA. It is likely that the insertion of the flipping-out base into the cavity in front of E396’ and the insertion of E488’ residue into the space vacated by flipping-out base take place in a brisk, concerted pace initially. Then, K594’ moves 4.5 Å away from E488’ (see Fig. 1 b) to contact the complementary RNA strand. That is, E488’ and K594’ work as two anchors to contact the substrate RNA strand (from the minor groove) and the complementary RNA strand (from the major groove), respectively, to stabilize the dsRNA. To characterize the ADAR1 conformational differences among the WT and the two studied mutants, we sorted 10,000 conformations evenly sampled over the last 1 µs of each trajectory. These conformations were arranged on a free energy landscape plotted against the E912-E1008 (as the y-axis) and E1008-K1120 (as the x-axis) separation distances (see Fig. 2 ). These distances were measured using the Cδ atom in the glutamate residue and the Cζ atom in the lysine residue. The 3’-binding loop II appears to be highly flexible, as the observed E1008-K1120 separation distance in wide range among the modeled systems. In the case of WT HM (see Fig. 2 a), it shows two conformation clusters. In cluster A, the E912-E1008 distance populates within 18 ± 1 Å (similar to the E396’-E488’ distance in the RNA-bound ADAR2 structure), and the E1008-K1120 distance populates within 9 ± 3 Å (similar to the E488’-K594’ distance in the apo ADAR2 structure). In cluster B, the E912-E1008 distance populates within 13 ± 1 Å (similar to the E396’-E488’ distance in the apo ADAR2 structure), and the E1008-K1120 distance populates within 16 ± 4 Å (similar to the E488’-K594’ distance in the RNA-bound ADAR2 structure). With regard to WT AF (see Fig. 2 b), the conformational entropy is greater than that of WT HM , and the center of the basin is at 18 Å of E912-E1008 distance and 15 Å of E1008-K1120 distance, which is similar to the RNA-bound ADAR2 structure. The relatively restricted distribution of the WT HM conformation compared to the WT AF model can be attributed to the template structure used during model construction. In the case of R892H HM (see Fig. 2 c), a basin with an E1008-K1120 distance of around 3 Å suggests the presence of an E1008-K1120 salt bridge. A significant portion of conformations in R892H AF (see Fig. 2 d), Y1112F HM (see Fig. 2 e), and Y1112F AF (see Fig. 2 f) exhibit an E1008-K1120 distance larger than 15 Å, suggesting that the 3’-binding loop II is pulled apart from the active site. Conversely, the relative position between E912 and E1008 in the studied systems is rather confined and mostly populates within 15 and 20 Å that are before and after the insertion of the flipping-out base observed in ADAR2. 3.3 Features in RNA-bound ADAR1 Structure We also constructed an RNA-bound ADAR1 model by including a dsRNA substrate into a WT AF conformation at 1.6 µs, with E912-E1008 (approximately 18 Å) and E1008-K1120 (approximately 15 Å) distances very similar to the E396’-E488’ and E488’-K594’ distances in the RNA-bound ADAR2 structure. Figure 3 displays a landscape of the 10,000 evenly sampled conformations from the entire 1 µs trajectory, organized over the same coordinates as in Fig. 2 . It is evident that ADAR1 is constrained by the bound RNA, as indicated by the narrow population of 14.8–15.3 Å for the E912-E1008 separation distance and 10–11 Å for the E1008-K1120 separation distance. As shown in Fig. 4 a, our modeled RNA-bound ADAR1 indicates that K1120 approaches the substrate RNA strand. This is in contrast to the corresponding K594’ that approaches the complementary RNA strand in ADAR2 (Fig. 1 b). Sequence alignment shows that ADAR1’s 3’-binding loop II (residues V1109-E1123) has one more residue (K1115) compared to ADAR2’s 3’-binding loop II (residues I584-N597), as indicated in Figure S1 . This causes ADAR1 K1120 to protrude toward the E1008 side and stabilize the substrate RNA strand, instead of the complementary RNA strand. Moreover, in the RNA-bound ADAR2 structure, a salt bridge formed by E588’ (in the 3’-binding loop II ) and R349’ (near the N-terminus of the β1 strand) is identified to stabilize the N-terminal end of the 3’-binding loop II (Fig. 1 c). Although ADAR2 E588’ and R349’ are conserved by ADAR1 D1113 and R866, an expected D1113-R866 salt bridge is not observed in our modeled structures (Fig. 4 b), possibly due to the shorter aspartate residue of D1113 compared to the glutamate residue of E588’. Lacking this salt bridge, ADAR1's 3’-binding loop II inherently exhibits flexibility, which is reflected in the broad distribution of E1008-K1120 separation distances in the six landscapes shown in Fig. 2 . 3.4 Structural Impact by R892H Our modeled WT HM and WT AF structures show R892 is able to link E1005 (on the base-flipping loop) and E1123 (the last residue of the flexible 3’-binding loop II ) to maintain the active site conformation. To corroborate this, we measure R892 (Cζ atom)-E1005 (Cδ atom) and R892-E1123 separation distances (Fig. 5 ). In WT HM (Fig. 5 a), R892 and E1123 are mostly approximately 2 Å apart, indicating the formation of a firm salt bridge. As for the distance between R892 and E1005, a portion of the population falls around 2 Å, while a significant portion falls between 6 and 8 Å, suggesting that the salt bridge between them is intermittently forming. The same R892-E1005 and R892-E1123 distance patterns were also observed in WT AF trajectory (Fig. 5 b). As for the R892H HM trajectory (Fig. 5 c), the substituted histidine residue is shorter than arginine, and as a result, H892 is unable to keep E1005 and E1123 close. This is evidenced by the H892 (Hε2 atom)-E1005 distance, mainly populated at 9 Å, and the H892-E1123 distance, mainly populated at 5 Å. Another significant structural aspect is the shorter N1006 (Cγ atom)-E1123 distance observed in Fig. 5 c, in comparison to the other three panels in Fig. 5 . A representative structure from the R892H HM trajectory (Fig. 6 a) illustrates how the outward-pointing H892 creates space for N1006 and E1123 to come into contact. The N1006-E1123 linkage subsequently triggers the E1008-K1120 connection, and this is supported by the R892H HM landscape (Fig. 2 c), where a great portion of the conformations are clustered with E1008-K1120 distance shorter than 5Å. Hence, the coalescence of flipping-base loop and 3’-binding loop II certainly averts the substrate dsRNA binding. The pattern of H892-E1005, H892-E1123, N1006-E1123 distance population in R892H AF (Fig. 5 d) is very similar to that of WT AF (Fig. 5 b). This similarity agrees with the similarity of the WT AF and R892F AF landscapes (Figs. 2 b and 2 d), suggesting high flexibility of the 3’-binding loop II . A represented structure retrieved from the R892H AF trajectory (see Fig. 6 b) shows K1120 sits away from the flipping-base loop; thus, it is impossible for K1120 to insert into the dsRNA’s groove. 3.5 Structural Impact by Y1112F The 3’-binding loop II , spanning from S1110 to E1123, can be divided into two segments with a pivotal point at G1119. The N-terminal half including S1110, I1111, Y1112, D1113, S1114, K1115, R1116, Q1117, and S1118 is underneath the α1 helix. The C-terminal half including G1119, K1120, T1121, K1122, and E1123 is flanking the active site and is responsible for stabilizing the substrate dsRNA. Accordingly, any mutation along the 3’-binding loop II reorients the C-terminal half and influences dsRNA recognition. Phenylalanine and tyrosine share a hydrophobic aromatic ring, with the sole structural distinction being the presence of a hydroxyl group in tyrosine. The solvent accessible surface areas (SASAs) for phenylalanine and tyrosine 314 Ų and 340 Ų, respectively, estimated by a 1.4 Å radius sphere. Consequently, we proceeded to compare their SASA percentages by taking the ratio of the exposed surface area to the total exposed SASA. As the violin plots in Fig. 7 , both WT HM and Y1112F HM have similar SASA% centering at 25% whereas WT AF and Y1112F AF have dramatically different SASA% values. To be more specific, in Y1112F AF the exposed F1112 area is between 5 to 25% whereas one third of the collected WT AF conformations expose Y1112 area up to 60%. As indicated in Figs. 8 a and 8 b, the embedded F1112 into the cleft enveloped by the N-terminal half of the 3’-binding loop II and the α1 helix compresses the F1112 surrounding and ultimately pulls the C-terminal half away from the flipping-base loop. To characterize the regional contraction introduced by Y1112F, we measure the separation distance between α1 helix’s H849 (the centroid of the imidazole ring) and 3’-binding lo loop II ’s S1114 (Cα atom). We also combined this with the E1008-K1120 separation distance to create six landscapes, which help categorize the collected conformations (see Fig. 9 ). In Figs. 9 a and 9 b, where a 10 Å H849-S1114 threshold was employed, a portion of the WT HM trajectory and the entirety of the WT AF trajectories are situated above this threshold, with an acceptable E1008-K1120 distance. In contrast, for Y1112F HM and Y1112F AF (Figs. 9 e and 9 f), the basins are located below the H849-S1114 threshold and feature longer E1008-K1120 distances. Figures 9 c and 9 d, depicting R892H, are included for reference to illustrate that the substitution of H892 does not drive regional contraction of the 3’-binding loop II and α1 helix. 4. Conclusion We explored ADAR1 deaminase structure and proposed criteria essential to efficient dsRNA accommodation. R892 is responsible for maintaining the positions of E1005 and E1123, thereby preserving the proper relative arrangement of the flipping-base loop and the 3’-binding loop II (see Fig. 10 a). As summarized in Figs. 10 b, the substitution of the smaller H892 disrupts this structural balance, resulting in either the shortened or elongated E1008-K1120 separation distance. This reasoning could also explain another point mutation responsible for Aicardi-Goutières syndrome, G1007R [ 35 ], where the substituted R1007 may experience electrostatic repulsion from R892, leading to an expanded E1008-K1120 distance that cannot effectively anchor the substrate dsRNA. The Y1112F structural influence is allosteric. Since the 3’-binding loop II is long and plastic, the presence of the substituted F1112 causes the 3’-binding loop II to shift toward the α1 helix, consequently moving K1120 away from the catalytic site, as depicted in Fig. 10 c. Declarations Declaration of competing interest The authors declare no competing financial interests or personal relationships that could influence the work reported in this study. Author Contribution C.N.Y. and W.C.H. performed the research, analyzed the data, wrote the paper and C.H.H. and T.A. participated in discussion. All authors reviewed the manuscript. Acknowledgement The authors thank Zuoying Armed Forces General Hospital, Kaohsiung, Taiwan (grant number: ZBH 108-04), for the financial support. CRediT authorship contribution statement Wen-Chieh Huang: performed the research, analyzed the data, wrote the paper. Chia-Hung Hsu: designed the research, participated in discussion. Titus Albu: participated in discussion. 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Biochemistry 57(10):1640–1651 Matthews MM, Thomas JM, Zheng Y et al (2016) Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat Struct Mol Biol 23(5):426–433 Macbeth MR, Schubert HL, Vandemark AP, Lingam AT, Hill CP, Bass BL (2005) Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309(5740):1534–1539 Matthews MM, Thomas JM, Zheng Y et al (2016) Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat Struct Mol Biol 23(5):426–433 Yu H, Bai K, Cheng Y et al (2023) Clinical significance, tumor immune landscape and immunotherapy responses of ADAR in pan-cancer and its association with proliferation and metastasis of bladder cancer. Aging 15(13):6302–6330 Bhate A, Sun T, Li JB (2019) ADAR1: A New Target for Immuno-oncology Therapy. Mol Cell 73(5):866–868 Ishizuka JJ, Manguso RT, Cheruiyot CK et al (2019) Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature 565(7737):43–48 Kung CP, Cottrell KA, Ryu S et al (2021) Evaluating the therapeutic potential of ADAR1 inhibition for triple-negative breast cancer. Oncogene 40(1):189–202 Binothman N, Aljadani M, Alghanem B et al (2023) Identification of novel interacts partners of ADAR1 enzyme mediating the oncogenic process in aggressive breast cancer. Sci Rep 13(1):8341 BIOVIA, Systèmes D (2020) Discovery Studio , San Diego: Dassault Systèmes, 2023 Hsu CH, Chen YJ, Yang CN (2022) An order-to-disorder structural switch regulates HIF-1 transcription through S247 phosphorylation in the HIF1α PAS-B domain. Comput Biol Med 149:106006 Chen YJ, Li PY, Yang CN (2021) Molecular dynamics study of enhanced autophosphorylation by S904F mutation of the RET kinase domain. J Struct Biol 213(4):107799 Chuang YC, Huang BY, Chang HW, Yang CN (2019) Molecular Modeling of ALK L1198F and/or G1202R Mutations to Determine Differential Crizotinib Sensitivity. Sci Rep 9(1):11390 Fisher AJ, Beal PA (2017) Effects of Aicardi-Goutières syndrome mutations predicted from ADAR-RNA structures. RNA Biol 14(2):164–170 Additional Declarations No competing interests reported. Supplementary Files ADAR1supplementary2024410.docx Cite Share Download PDF Status: Published Journal Publication published 17 Jul, 2024 Read the published version in Journal of Computer-Aided Molecular Design → Version 1 posted Reviews received at journal 21 Jun, 2024 Reviewers agreed at journal 21 Jun, 2024 Reviewers invited by journal 21 May, 2024 Editor assigned by journal 21 May, 2024 Submission checks completed at journal 03 May, 2024 First submitted to journal 01 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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Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chia-Hung","middleName":"","lastName":"Hsu","suffix":""},{"id":298203965,"identity":"9ee2636c-1dac-4946-8a11-50810a2535b3","order_by":2,"name":"Titus Albu","email":"","orcid":"","institution":"University of Tennessee","correspondingAuthor":false,"prefix":"","firstName":"Titus","middleName":"","lastName":"Albu","suffix":""},{"id":298203967,"identity":"6c314de0-e9c3-41c1-befb-d6fb2cd1c56f","order_by":3,"name":"Chia-Ning Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYPACGwYGZuYGOFeCCC1pQC2MKFoMCGk5DMTEauGf3WP4ueDX+Wj+dsYG5oI/h+0NDjAfvM3D8CexAYcWiTtnjKVn9t3OnXEYqGVm2+HEDQfYkq15GAxwajGQyDGQ5u25ndsA0sLbcDjB4ACPmTRQSy4eLca/eXvO5c4HaeEBO4z/GyEtQDN/HMjdANbCdphxwwEeNrxaJG6klVnzNiTnbgRqOczblp448zCbseUcA+N6XFr4ZyRvvs3zxy533vnDBx/z/LG25zve/PDGmwo5Yxw6gIDDgIGxDcI8wMDQDIxTsINxa2BgYH/AwPAHzqvDp3QUjIJRMApGKAAAXw9XDJIYWLkAAAAASUVORK5CYII=","orcid":"","institution":"National Sun Yat-sen University","correspondingAuthor":true,"prefix":"","firstName":"Chia-Ning","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-05-02 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8","display":"","copyAsset":false,"role":"figure","size":497813,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4356501/v1/84655b8c8dc9a9ab5c88e0dc.jpg"},{"id":56177004,"identity":"0d84d33c-7504-40f6-8c75-5fd3c1929914","added_by":"auto","created_at":"2024-05-09 13:18:51","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":438121,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4356501/v1/14294a8fe5a59f122506cd2d.jpg"},{"id":56176974,"identity":"c7ac26c8-98c4-4a30-8e8b-f0232ee6dc05","added_by":"auto","created_at":"2024-05-09 13:18:46","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":385456,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4356501/v1/316b3bf631ca2a07ad5a111d.jpg"},{"id":61595133,"identity":"b4b38635-c302-42b1-bdb1-6ca6675c76cd","added_by":"auto","created_at":"2024-08-01 17:20:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4374191,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4356501/v1/60e9bea8-aa46-4375-9ebc-132131449470.pdf"},{"id":56177543,"identity":"16bfa7c2-80a7-45c8-ab45-5d653a5b8f85","added_by":"auto","created_at":"2024-05-09 13:26:54","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":2912270,"visible":true,"origin":"","legend":"","description":"","filename":"ADAR1supplementary2024410.docx","url":"https://assets-eu.researchsquare.com/files/rs-4356501/v1/df0a8b8d8749e994828e8841.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structural Impacts of Two Disease-linked ADAR1 Mutants: A Molecular Dynamics Study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAdenosine deaminase acting on RNA (ADAR) is a class of enzymes to process the adenosine-to-inosine (A-to-I) RNA editing through deamination reaction, making it the most prevalent type of post-transcriptional nucleotide modification in humans [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Since inosine can form Watson-Crick base pairing with cytidine, the resulted inosine behaves like guanosine and, as a result, the A-to-I editing on RNA significantly modifies RNA function, such as miRNA biosynthesis and target recognition, splicing patterns, gene regulation, and the long-non-coding RNA functions [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are three ADARs in humans, namely ADAR1, ADAR2, and ADAR3 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Both ADAR1 and ADAR2 are catalytically active and expressed in almost all tissues, whereas ADAR3 is only expressed in brain and has no detectable catalytic activity. It has been reported that ADAR3 can compete with ADAR2 to inhibit RNA editing by directing its binding to GRIA2 pre-mRNA in glioblastoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Because ADAR1\u0026rsquo;s editing functions on dsRNA inhibits innate immunity and the interferon-mediated response, dysregulation of ADAR1 activity causes upregulated interferon and autoinflammatory diseases such as Aicardi-Goutieres syndrome (AGS) and dyschromatosis symmetrica hereditarian (DSH) [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. AGS is a rare childhood autoimmune disorder that primarily affects the brain, immune system, and skin. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] Currently it has been reported that seven genes, including ADAR1, confirmed to be associated with AGS [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The severity of the disease caused by the loss-of-function ADAR mutations may depend on the overall impact of these mutations on A-to-I RNA editing activity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A decrease in editing activity below a certain threshold likely triggers the development of a neurological disorder [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMolecular dynamics (MD) simulations have long been a useful tool in providing structural and dynamic details of proteins [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this work, we carried out MD simulations to study two AGS-causing mutations in ADAR1 deaminase domain, R892H and Y1112F, identifying their structural deviation from the wild-type (WT). From the N-terminus to the C-terminus, ADAR1 includes two Z-DNA binding domains (ZBDs), three dsRNA binding domains, and deaminase domain. Currently, the solved structure of ADAR1 is limited to the ZBDs [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We used homology modeling to build one apo ADAR1 deaminase domain, employing the solved human ADAR2 deaminase domain structure as a template. To enhance our conformational sampling, we also generated another ADAR1 structure using AlphaFold2, a method known for its high precision in protein structure prediction [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For each of the two model structures, the R892 residue was replaced by histadine to form two R892H strucures, and the Y1112 residue was replaced by phenealanine to form two Y1112F strucures. All of the six structures underwent MD simulations to refine and collect conformations, ultimately helping us deduce the structural criteria for RNA substrate recognition. Additionally, we retrieved a wild-type conformation from the AlphaFold2 trajectory, superimposed it with the RNA-bound ADAR2 structure, replaced the RNA sequence by HER1 RNA [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and submitted it for MD simulations.\u003c/p\u003e \u003cp\u003eSequence alignment of ADAR1 and ADAR2 deaminase domains (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) indicates 39% sequence identity and 59% sequence similarity, suggesting that the available ADAR2 structural data provides useful guidance in envisioning how ADAR1 associates with a dsRNA substrate. The ADAR2 deaminase domain is composed of 20 loops, four of which come into contact with the dsRNA. As indicated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, these loops are referred to as the base-flipping loop (E485\u0026rsquo;-G489\u0026rsquo;), the 5'-binding loop (A454\u0026rsquo;-R477\u0026rsquo;), the 3'-binding loop\u003csup\u003eI\u003c/sup\u003e (A347\u0026rsquo;-L352\u0026rsquo;), and the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e (L584\u0026rsquo;-N597\u0026rsquo;) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For ease of reading, ARAD2 residues are marked with primes to distinguish them from the unprimed ADAR1 residues. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, ADAR2 catalytic site is enclosed by E396\u0026rsquo;, E488\u0026rsquo;, and K594. The E396\u0026rsquo; (on the N-terminal end of α2 helix in the core structure) secures the flipping-out adenine (on the substrate RNA strand) base while the E488\u0026rsquo; (on the base-flipping loop) inserts into the minor groove, filling the space left by the flipping-out adenine, and contacts the unpaired, orphaned base located on the complementary RNA strand. Taking the substrate RNA strand as a viewpoint, from top to bottom in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, the 3\u0026rsquo; end and 5\u0026rsquo; end regions relative to the flipping-out base, which is to be edited, are in contact with the 3\u0026rsquo;-binding loops and 5\u0026rsquo;-binding loop, respectively. Because of its high flexibility, the 24-base-long 5\u0026rsquo;-binding loop structure was not resolved in the apo ADAR2 structure (PDB: 1ZY7) but was resolved in the RNA-ADAR2 complex (PDB: 5HP2) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The R348\u0026rsquo; (in the 3\u0026rsquo;-binding loop\u003csup\u003eI\u003c/sup\u003e) and K594\u0026rsquo; (in the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e) both stabilize the dsRNA. Except R348\u0026rsquo; not conserved by ADAR1 G865, the catalytic site\u0026rsquo;s E396\u0026rsquo;, E488\u0026rsquo;, and K594\u0026rsquo; are conserved by ADAR1\u0026rsquo;s E912, E1008, and K1120. The E912-E1008 and E1008-K1120 separation distances were adopted to categorize the collected WT, R892H, and Y1112F conformations. We hypothesized that if the E1008-K1120 distance exceeds the tolerance level, it would discourage the dsRNA recognition process, ultimately reducing the A-to-I editing activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNumerous AGS-causing mutations are on the ADAR1 deaminase domain, including A870T (A353\u0026rsquo; in ADAR2), I872T (V355\u0026rsquo;), R892H (K376\u0026rsquo;), K999N (Q479\u0026rsquo;), G1007R (G487\u0026rsquo;), Y1112F (A587\u0026rsquo;), and D1113H (E588\u0026rsquo;), as summarized in Figure S2. By analyzing these corresponding residues on the ADAR2 deaminase domain structure, we foresee the possible consequence of ADAR1\u0026rsquo;s mutations. For example, A353\u0026rsquo; and V355\u0026rsquo; are in vicinity of E396\u0026rsquo; in ADAR2 and accordingly either A870T or I872T mutation impairs the interaction between E912 and the flipping base in ADAR1. Because G487\u0026rsquo; is next to E488\u0026rsquo; and K376\u0026rsquo; holds salt bridges to secure the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e, either G1007R or R896H mutation surely deforms the active site. Q479\u0026rsquo; is in the C-terminus of the 5\u0026rsquo;-binding loop and thus the correlated K999N mutation may interfere the induced-fit process when associating the dsRNA substrate. A587\u0026rsquo;, E588\u0026rsquo;, and K594\u0026rsquo; are located in the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e; thus Y1112F and D1113H mutations may pull K1120 away from the active site. Since ADAR1\u0026rsquo;s Y1112 and ADAR2\u0026rsquo;s A587\u0026rsquo; possess dissimilar sizes and hydrophilicities, it is necessary to learn the role played by Y1112 before investigating Y1112F mutation. Our objective is to explore the susceptibility of ADAR1's active site. To achieve this, we selected an orthosteric site R892H mutation and an allosteric site Y1112F mutation. Our conformational analysis suggests that in R892H variant, the substituted H892 is not able to maintain the R892-E1123 ionic linkage. On the other hand, the hydrophobic F1112 in Y1112F mutant reduces its solvent-accessible surface area (SASA) and causes a contraction movement of the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e toward the α1 helix. Both mutations rearrange K1120 position and interfere the substrate RNA recognition.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 ADAR1 Structure Preparation\u003c/h2\u003e \u003cp\u003eWe used the AMBER 18 software package for MD simulations. To build the initial WT structure, we employed the homology modeling module in Discovery Studio 2020 program [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], using the ADAR2 deaminase domain structure (PDB: 1ZY7) as a template [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This choice was supported by their 39% sequence identity and 59% sequence similarity. (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). It's important to note that the success of homology modeling in predicting protein structure greatly depends on sequence identity, with a typical criterion being around 30%. In addition, we assessed the model's accuracy and evaluated the correctness of the model prediction using the PROCHECK scoring algorithm, which enables a more precise evaluation of the reliability of the modeling results. To intensify our conformational sampling, a second prediction was carried out with AlphaFold2 method without assigning any template. We further compared the two predicted ADAR1 structures with the crystallography-solved apo ADAR2, excluding the 5'-binding loop, which was not resolved in the apo ADAR2 structure. This loop was observed in the ADAR2-RNA complex, suggesting an induced-fit conformational change upon substrate binding in ADAR2. The root-mean-square deviation (RMSD) calculated between the homology-built ADAR1 and the solved ADAR2 is 1.963, between the AlphaFold2-built ADAR1 and the solved ADAR2 is 4.446, and between the homology-built ADAR1 and AlphaFold2 ADAR1 is 4.691. Superpositions among the three structures (see Figures S3a-c) indicate that the deviations are primarily in the loops flanking the structure core. Since the flexible 5\u0026rsquo;-binding loop is expected to remain freely exposed to the solvent, we replaced all 24 amino acid residues with 24 sequential alanine residues and surrounded them with water molecules. This modification was made to keep the loop away from the core structure and minimize any structural biases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 ADAR1-RNA Complex Structure Preparation\u003c/h2\u003e \u003cp\u003eTo investigate the engagement between our ADAR1 model and a dsRNA substrate in the catalytic site, we constructed an RNA-bound ADAR1 model by taking a frame from the AlphaFold2 trajectory at 1.6 \u0026micro;s due to its remarkable conformational similarity to the RNA-bound form of the ADAR2 deaminase. We superimposed it to the RNA-ADAR2 complex structure (PDB: 5HP2) and substituted the bound RNA sequence with a short segment of the HER1 RNA sequence [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Initially we constrained the RNA structure while allowing the ADAR1 structure to adapt to accommodate the substrate, and later we relaxed the entire complex to sample conformations. Since the simulation duration was short, the induced-fit process of the 5\u0026rsquo;-binding loop was not considered.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Molecular Dynamics Simulations\u003c/h2\u003e \u003cp\u003eEach studied structure was immersed in a cubic TIP3P water box, with the walls maintained at least 10 \u0026Aring; away from the immersed ADAR1 structure. The energy minimization of each solvated system was conducted in three stages, as in our previous studies [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The Particle-Mesh Ewald method was applied to handle long-range electrostatic interactions, and the SHAKE algorithm was used to constrain all bonds containing hydrogen atoms to their equilibrium lengths. An 8 \u0026Aring; cutoff distance was set for efficient simulation of nonbonding interactions such as short-range electrostatics and van der Waals interactions. Each simulated ADAR1 WT structure underwent a gradual heating process from 0 to 300 K over 100 ps, followed by a density equilibration procedure at the target temperature for 100 ps, and then constant equilibration at the target temperature for 1000 ps. Subsequently, the systems were subjected to a 2 \u0026micro;s simulation run. In the case of the two mutants, we selected a WT conformation at the 1 \u0026micro;s time frame and replaced R892 with histidine and Y1112 with phenylalanine to create the initial structures of the R892H and Y1112F variants, respectively. To maintain the electroneutrality of the studied systems, we added seven chloride ions to the WT and Y1112F simulations, six chloride ions to the R892H simulation, and 27 sodium ions to the ADAR1-RNA complex simulation. The setup and protocols mentioned above for WT\u0026rsquo;s heating, density equilibration, and equilibrium were also applied to process the four mutant trajectories and one RNA-bound complex trajectory. In the following these six trajectories are termed WT\u003csup\u003eHM\u003c/sup\u003e, WT\u003csup\u003eAF\u003c/sup\u003e, R892H\u003csup\u003eHM\u003c/sup\u003e, R892H\u003csup\u003eAF\u003c/sup\u003e, Y1112F\u003csup\u003eHM\u003c/sup\u003e, and Y1112F\u003csup\u003eAF\u003c/sup\u003e, based on the conducted homology modeling (HM) and AlphaFold2 (AF) approach. The length for each of the six apo ADAR1 trajectories is 2 \u0026micro;s and for the RNA-bound ADAR1 is 1 \u0026micro;s.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result and Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 MD Stability\u003c/h2\u003e \u003cp\u003eEach of the WT, R892H variant, and Y1112F variant was simulated with two independent replicas for conformation collection. As indicated in Figure S4, RMSF (root mean square fluctuation) values plotted against amino acid residues show high flexibility in the 5\u0026rsquo; end binding loop (residues 972\u0026ndash;995), which was replaced by 24 alanine residues in each of the six trajectories. Consequently, the coordinates of the 5\u0026rsquo; end binding loop were excluded from the RMSD (root mean square deviation) calculation. Our RMSD calculation was based on the heavy atoms throughout the entire 2 \u0026micro;s production duration. All the modeling systems show a rapidly increasing RMSD values and a deviation from their initial structure of approximately 3 \u0026Aring; (see Figure S5). As indicated in Figure S5a the WT\u003csup\u003eHM\u003c/sup\u003e trajectory reached 3.0 \u0026Aring; around 800 ns and maintained a stable centering at 3.2 \u0026Aring; between 800 and 2000 ns. Likely, in Figure S5b, the second half of the WT\u003csup\u003eAF\u003c/sup\u003e trajectory is stable, centering at 3.5 \u0026Aring;. The two R892H\u003csup\u003eHM\u003c/sup\u003e and R892H\u003csup\u003eAF\u003c/sup\u003e trajectories exhibit higher variation, reaching nearly 4 \u0026Aring; after 1100 ns from their initial structures (see Figure S5c and S5d). The Y1112F\u003csup\u003eHM\u003c/sup\u003e trajectory became stable around 1000 ns (see Figure S5e), whereas the Y1112F\u003csup\u003eAF\u003c/sup\u003e curve maintained a mean value of 3.4 \u0026Aring; between 900 and 1300 ns and increased to a mean value of 3.8 \u0026Aring; between 1300 and 2000 ns (see Figure S5f), implying a regional structural change along the trajectory. Following this, we captured snapshots from each trajectory, ranging from 1 \u0026micro;s to 2 \u0026micro;s, for structural investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Conformational Discrepancy among the Variants\u003c/h2\u003e \u003cp\u003eMeasured between the Cδ atom in the glutamate residue and the Cζ atom in the lysine residue, the E396\u0026rsquo;-E488\u0026rsquo; separation distance is 14.6 \u0026Aring; in the apo ADAR2 structure and is 18 \u0026Aring; in the RNA-bound ADAR2 structure; the E488\u0026rsquo;-K594\u0026rsquo; separation distance is 9.4 \u0026Aring; in the apo structure and is 15.1 \u0026Aring; in the RNA-bound structure. The condensed apo ADAR2 conformation could be attributed to the salt condition during crystallization or its intrinsic nature. Apparently, the apo ADAR2 conformation undergoes induced-fit relaxation when accommodating the dsRNA. It is likely that the insertion of the flipping-out base into the cavity in front of E396\u0026rsquo; and the insertion of E488\u0026rsquo; residue into the space vacated by flipping-out base take place in a brisk, concerted pace initially. Then, K594\u0026rsquo; moves 4.5 \u0026Aring; away from E488\u0026rsquo; (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) to contact the complementary RNA strand. That is, E488\u0026rsquo; and K594\u0026rsquo; work as two anchors to contact the substrate RNA strand (from the minor groove) and the complementary RNA strand (from the major groove), respectively, to stabilize the dsRNA.\u003c/p\u003e \u003cp\u003eTo characterize the ADAR1 conformational differences among the WT and the two studied mutants, we sorted 10,000 conformations evenly sampled over the last 1 \u0026micro;s of each trajectory. These conformations were arranged on a free energy landscape plotted against the E912-E1008 (as the y-axis) and E1008-K1120 (as the x-axis) separation distances (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These distances were measured using the Cδ atom in the glutamate residue and the Cζ atom in the lysine residue. The 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e appears to be highly flexible, as the observed E1008-K1120 separation distance in wide range among the modeled systems. In the case of WT\u003csup\u003eHM\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), it shows two conformation clusters. In cluster A, the E912-E1008 distance populates within 18\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026Aring; (similar to the E396\u0026rsquo;-E488\u0026rsquo; distance in the RNA-bound ADAR2 structure), and the E1008-K1120 distance populates within 9\u0026thinsp;\u0026plusmn;\u0026thinsp;3 \u0026Aring; (similar to the E488\u0026rsquo;-K594\u0026rsquo; distance in the apo ADAR2 structure). In cluster B, the E912-E1008 distance populates within 13\u0026thinsp;\u0026plusmn;\u0026thinsp;1 \u0026Aring; (similar to the E396\u0026rsquo;-E488\u0026rsquo; distance in the apo ADAR2 structure), and the E1008-K1120 distance populates within 16\u0026thinsp;\u0026plusmn;\u0026thinsp;4 \u0026Aring; (similar to the E488\u0026rsquo;-K594\u0026rsquo; distance in the RNA-bound ADAR2 structure). With regard to WT\u003csup\u003eAF\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), the conformational entropy is greater than that of WT\u003csup\u003eHM\u003c/sup\u003e, and the center of the basin is at 18 \u0026Aring; of E912-E1008 distance and 15 \u0026Aring; of E1008-K1120 distance, which is similar to the RNA-bound ADAR2 structure. The relatively restricted distribution of the WT\u003csup\u003eHM\u003c/sup\u003e conformation compared to the WT\u003csup\u003eAF\u003c/sup\u003e model can be attributed to the template structure used during model construction. In the case of R892H\u003csup\u003eHM\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), a basin with an E1008-K1120 distance of around 3 \u0026Aring; suggests the presence of an E1008-K1120 salt bridge. A significant portion of conformations in R892H\u003csup\u003eAF\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), Y1112F\u003csup\u003eHM\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), and Y1112F\u003csup\u003eAF\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef) exhibit an E1008-K1120 distance larger than 15 \u0026Aring;, suggesting that the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e is pulled apart from the active site. Conversely, the relative position between E912 and E1008 in the studied systems is rather confined and mostly populates within 15 and 20 \u0026Aring; that are before and after the insertion of the flipping-out base observed in ADAR2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Features in RNA-bound ADAR1 Structure\u003c/h2\u003e \u003cp\u003eWe also constructed an RNA-bound ADAR1 model by including a dsRNA substrate into a WT\u003csup\u003eAF\u003c/sup\u003e conformation at 1.6 \u0026micro;s, with E912-E1008 (approximately 18 \u0026Aring;) and E1008-K1120 (approximately 15 \u0026Aring;) distances very similar to the E396\u0026rsquo;-E488\u0026rsquo; and E488\u0026rsquo;-K594\u0026rsquo; distances in the RNA-bound ADAR2 structure. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays a landscape of the 10,000 evenly sampled conformations from the entire 1 \u0026micro;s trajectory, organized over the same coordinates as in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. It is evident that ADAR1 is constrained by the bound RNA, as indicated by the narrow population of 14.8\u0026ndash;15.3 \u0026Aring; for the E912-E1008 separation distance and 10\u0026ndash;11 \u0026Aring; for the E1008-K1120 separation distance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, our modeled RNA-bound ADAR1 indicates that K1120 approaches the substrate RNA strand. This is in contrast to the corresponding K594\u0026rsquo; that approaches the complementary RNA strand in ADAR2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Sequence alignment shows that ADAR1\u0026rsquo;s 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e (residues V1109-E1123) has one more residue (K1115) compared to ADAR2\u0026rsquo;s 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e (residues I584-N597), as indicated in Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. This causes ADAR1 K1120 to protrude toward the E1008 side and stabilize the substrate RNA strand, instead of the complementary RNA strand. Moreover, in the RNA-bound ADAR2 structure, a salt bridge formed by E588\u0026rsquo; (in the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e) and R349\u0026rsquo; (near the N-terminus of the β1 strand) is identified to stabilize the N-terminal end of the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Although ADAR2 E588\u0026rsquo; and R349\u0026rsquo; are conserved by ADAR1 D1113 and R866, an expected D1113-R866 salt bridge is not observed in our modeled structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), possibly due to the shorter aspartate residue of D1113 compared to the glutamate residue of E588\u0026rsquo;. Lacking this salt bridge, ADAR1's 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e inherently exhibits flexibility, which is reflected in the broad distribution of E1008-K1120 separation distances in the six landscapes shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Structural Impact by R892H\u003c/h2\u003e \u003cp\u003eOur modeled WT\u003csup\u003eHM\u003c/sup\u003e and WT\u003csup\u003eAF\u003c/sup\u003e structures show R892 is able to link E1005 (on the base-flipping loop) and E1123 (the last residue of the flexible 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e) to maintain the active site conformation. To corroborate this, we measure R892 (Cζ atom)-E1005 (Cδ atom) and R892-E1123 separation distances (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In WT\u003csup\u003eHM\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), R892 and E1123 are mostly approximately 2 \u0026Aring; apart, indicating the formation of a firm salt bridge. As for the distance between R892 and E1005, a portion of the population falls around 2 \u0026Aring;, while a significant portion falls between 6 and 8 \u0026Aring;, suggesting that the salt bridge between them is intermittently forming. The same R892-E1005 and R892-E1123 distance patterns were also observed in WT\u003csup\u003eAF\u003c/sup\u003e trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). As for the R892H\u003csup\u003eHM\u003c/sup\u003e trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), the substituted histidine residue is shorter than arginine, and as a result, H892 is unable to keep E1005 and E1123 close. This is evidenced by the H892 (Hε2 atom)-E1005 distance, mainly populated at 9 \u0026Aring;, and the H892-E1123 distance, mainly populated at 5 \u0026Aring;. Another significant structural aspect is the shorter N1006 (Cγ atom)-E1123 distance observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, in comparison to the other three panels in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. A representative structure from the R892H\u003csup\u003eHM\u003c/sup\u003e trajectory (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) illustrates how the outward-pointing H892 creates space for N1006 and E1123 to come into contact. The N1006-E1123 linkage subsequently triggers the E1008-K1120 connection, and this is supported by the R892H\u003csup\u003eHM\u003c/sup\u003e landscape (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), where a great portion of the conformations are clustered with E1008-K1120 distance shorter than 5\u0026Aring;. Hence, the coalescence of flipping-base loop and 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e certainly averts the substrate dsRNA binding.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe pattern of H892-E1005, H892-E1123, N1006-E1123 distance population in R892H\u003csup\u003eAF\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed) is very similar to that of WT\u003csup\u003eAF\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). This similarity agrees with the similarity of the WT\u003csup\u003eAF\u003c/sup\u003e and R892F\u003csup\u003eAF\u003c/sup\u003e landscapes (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), suggesting high flexibility of the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e. A represented structure retrieved from the R892H\u003csup\u003eAF\u003c/sup\u003e trajectory (see Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) shows K1120 sits away from the flipping-base loop; thus, it is impossible for K1120 to insert into the dsRNA\u0026rsquo;s groove.\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Structural Impact by Y1112F\u003c/h2\u003e \u003cp\u003eThe 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e, spanning from S1110 to E1123, can be divided into two segments with a pivotal point at G1119. The N-terminal half including S1110, I1111, Y1112, D1113, S1114, K1115, R1116, Q1117, and S1118 is underneath the α1 helix. The C-terminal half including G1119, K1120, T1121, K1122, and E1123 is flanking the active site and is responsible for stabilizing the substrate dsRNA. Accordingly, any mutation along the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e reorients the C-terminal half and influences dsRNA recognition. Phenylalanine and tyrosine share a hydrophobic aromatic ring, with the sole structural distinction being the presence of a hydroxyl group in tyrosine. The solvent accessible surface areas (SASAs) for phenylalanine and tyrosine 314 \u0026Aring;\u0026sup2; and 340 \u0026Aring;\u0026sup2;, respectively, estimated by a 1.4 \u0026Aring; radius sphere. Consequently, we proceeded to compare their SASA percentages by taking the ratio of the exposed surface area to the total exposed SASA. As the violin plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, both WT\u003csup\u003eHM\u003c/sup\u003e and Y1112F\u003csup\u003eHM\u003c/sup\u003e have similar SASA% centering at 25% whereas WT\u003csup\u003eAF\u003c/sup\u003e and Y1112F\u003csup\u003eAF\u003c/sup\u003e have dramatically different SASA% values. To be more specific, in Y1112F\u003csup\u003eAF\u003c/sup\u003e the exposed F1112 area is between 5 to 25% whereas one third of the collected WT\u003csup\u003eAF\u003c/sup\u003e conformations expose Y1112 area up to 60%. As indicated in Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb, the embedded F1112 into the cleft enveloped by the N-terminal half of the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e and the α1 helix compresses the F1112 surrounding and ultimately pulls the C-terminal half away from the flipping-base loop.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize the regional contraction introduced by Y1112F, we measure the separation distance between α1 helix\u0026rsquo;s H849 (the centroid of the imidazole ring) and 3\u0026rsquo;-binding lo loop\u003csup\u003eII\u003c/sup\u003e\u0026rsquo;s S1114 (Cα atom). We also combined this with the E1008-K1120 separation distance to create six landscapes, which help categorize the collected conformations (see Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb, where a 10 \u0026Aring; H849-S1114 threshold was employed, a portion of the WT\u003csup\u003eHM\u003c/sup\u003e trajectory and the entirety of the WT\u003csup\u003eAF\u003c/sup\u003e trajectories are situated above this threshold, with an acceptable E1008-K1120 distance. In contrast, for Y1112F\u003csup\u003eHM\u003c/sup\u003e and Y1112F\u003csup\u003eAF\u003c/sup\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ef), the basins are located below the H849-S1114 threshold and feature longer E1008-K1120 distances. Figures\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed, depicting R892H, are included for reference to illustrate that the substitution of H892 does not drive regional contraction of the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e and α1 helix.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e\u0026lt;Insert\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cb\u003e\u0026gt;\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eWe explored ADAR1 deaminase structure and proposed criteria essential to efficient dsRNA accommodation. R892 is responsible for maintaining the positions of E1005 and E1123, thereby preserving the proper relative arrangement of the flipping-base loop and the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e (see Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea). As summarized in Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb, the substitution of the smaller H892 disrupts this structural balance, resulting in either the shortened or elongated E1008-K1120 separation distance. This reasoning could also explain another point mutation responsible for Aicardi-Gouti\u0026egrave;res syndrome, G1007R [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], where the substituted R1007 may experience electrostatic repulsion from R892, leading to an expanded E1008-K1120 distance that cannot effectively anchor the substrate dsRNA. The Y1112F structural influence is allosteric. Since the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e is long and plastic, the presence of the substituted F1112 causes the 3\u0026rsquo;-binding loop\u003csup\u003eII\u003c/sup\u003e to shift toward the α1 helix, consequently moving K1120 away from the catalytic site, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ec.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing financial interests or personal relationships that could influence the work reported in this study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.N.Y. and W.C.H. performed the research, analyzed the data, wrote the paper and C.H.H. and T.A. participated in discussion. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors thank Zuoying Armed Forces General Hospital, Kaohsiung, Taiwan (grant number: ZBH 108-04), for the financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWen-Chieh Huang: performed the research, analyzed the data, wrote the paper.\u003c/p\u003e\n\u003cp\u003eChia-Hung Hsu: designed the research, participated in discussion.\u003c/p\u003e\n\u003cp\u003eTitus Albu: participated in discussion.\u003c/p\u003e\n\u003cp\u003eChia-Ning Yang: designed research, analyzed the data, wrote the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eThomas JM, Beal PA (2017) How do ADARs bind RNA? New protein-RNA structures illuminate substrate recognition by the RNA editing ADARs. 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RNA Biol 14(2):164\u0026ndash;170\u003c/span\u003e\u003c/li\u003e\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":"journal-of-computer-aided-molecular-design","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcam","sideBox":"Learn more about [Journal of Computer-Aided Molecular Design](http://link.springer.com/journal/10822)","snPcode":"10822","submissionUrl":"https://submission.nature.com/new-submission/10822/3","title":"Journal of Computer-Aided Molecular Design","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4356501/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4356501/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdenosine deaminases acting on RNA (ADARs) are pivotal RNA-editing enzymes responsible for converting adenosine to inosine within double-stranded RNA (dsRNA). Dysregulation of ADAR1 editing activity, often arising from genetic mutations, has been linked to elevated interferon levels and the onset of autoinflammatory diseases. However, understanding the molecular underpinnings of this dysregulation is impeded by the lack of an experimentally determined structure for the ADAR1 deaminase domain. In this computational study, we utilized homology modeling and the AlphaFold2 to construct structural models of the ADAR1 deaminase domain in wild-type and two pathogenic variants, R892H and Y1112F, to decipher the structural impact on the reduced deaminase activity. Our findings illuminate the critical role of structural complementarity between the ADAR1 deaminase domain and dsRNA in enzyme-substrate recognition. That is, the relative position of E1008 and K1120 must be maintained so that they can insert into the minor and major grooves of the substrate dsRNA, respectively, facilitating the flipping-out of adenosine to be accommodated within a cavity surrounding E912. Both the orthosteric R892 mutations of R892 and the allosteric Y1112F mutation alter K1120 position and ultimately hinder substrate RNA binding.\u003c/p\u003e","manuscriptTitle":"Structural Impacts of Two Disease-linked ADAR1 Mutants: A Molecular Dynamics Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-09 13:18:26","doi":"10.21203/rs.3.rs-4356501/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2024-06-21T16:23:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"297589533035637429171767232675634870131","date":"2024-06-21T16:21:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-21T18:03:28+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-21T18:02:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-03T04:11:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Computer-Aided Molecular Design","date":"2024-05-02T02:48:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-computer-aided-molecular-design","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcam","sideBox":"Learn more about [Journal of Computer-Aided Molecular Design](http://link.springer.com/journal/10822)","snPcode":"10822","submissionUrl":"https://submission.nature.com/new-submission/10822/3","title":"Journal of Computer-Aided Molecular Design","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"71ab751e-a158-42c7-a34d-f5e65c7a98e5","owner":[],"postedDate":"May 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T16:18:49+00:00","versionOfRecord":{"articleIdentity":"rs-4356501","link":"https://doi.org/10.1007/s10822-024-00565-1","journal":{"identity":"journal-of-computer-aided-molecular-design","isVorOnly":false,"title":"Journal of Computer-Aided Molecular Design"},"publishedOn":"2024-07-17 16:13:02","publishedOnDateReadable":"July 17th, 2024"},"versionCreatedAt":"2024-05-09 13:18:26","video":"","vorDoi":"10.1007/s10822-024-00565-1","vorDoiUrl":"https://doi.org/10.1007/s10822-024-00565-1","workflowStages":[]},"version":"v1","identity":"rs-4356501","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4356501","identity":"rs-4356501","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}