Structural and Biochemical Characterization of Grimontia hollisae Thermostable Direct Hemolysin with DNA Reveals First Vibrio Hemolysin with Nuclease Activity

Structural and Biochemical Characterization of Grimontia hollisae Thermostable Direct Hemolysin with DNA Reveals First Vibrio Hemolysin with Nuclease Activity | 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 Article Structural and Biochemical Characterization of Grimontia hollisae Thermostable Direct Hemolysin with DNA Reveals First Vibrio Hemolysin with Nuclease Activity Chin-Yuan Chang, Po-Yun Hsiao, Yu-Kuo Wang, Sheng-Cih Huang, Feng-Pai Chou, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5915599/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Grimontia hollisae thermostable direct hemolysin (Gh-TDH) is a pore-forming toxin that disrupts cell membrane, causing erythrocyte lysis and exhibiting cytotoxicity, cardiotoxicity, enterotoxicity, hepatotoxicity, and lethality in mice. Recombinant Gh-TDH-FITC binds to hepatocyte membranes and translocates to the nucleus. This study reveals that Gh-TDH cleaves DNA with 3'–5' nuclease activity to generate a 3-mer fragment, while cleaving RNA with ~ 70% lower efficiency. The co-crystal structure of Gh-TDH bound to ssDNA unveils a unique DNA-binding configuration, where eight ssDNA strands form four duplexes, each connected to a Gh-TDH tetramer at one end and to protomers from four different tetramers at the other. Notably, the cleavage site (Tyr87-Lys88-Asp89) deviates from the canonical 3'–5' exonuclease motif (Asp-Glu-Asp-Asp). Mutagenesis identified Lys88 as a general base essential for nuclease activity. This study provides structural and functional insights of Gh-TDH on DNA-binding and cleavage mechanism. In summary, Gh-TDH is the first pore-forming toxin identified in the Vibrio genus with dual functions of hemolytic and nuclease activities. Biological sciences/Biochemistry/DNA Biological sciences/Biochemistry/Structural biology/X-ray crystallography Biological sciences/Biochemistry/Enzymes Grimontia hollisae thermostable direct hemolysin pore-forming toxin exonuclease activity binding and internalization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pathogenic bacteria produce a range of toxins, including pore-forming toxins (PFTs), cytotoxins, neurotoxins, superantigens, and nucleases, to damage host cells and evade immune responses. For example, Vibrio cholerae cytolysin disrupts cell membranes, causing severe gastrointestinal symptoms, while Streptococcus pneumoniae DNase degrades neutrophil extracellular traps (NETs), aiding immune evasion 1–3 . Group A Streptococcus (GAS) DNase exacerbates disease by degrading host DNA, reducing pus viscosity, and facilitating bacterial spread 4 . Similarly, Mycoplasma bovis MbovNase degrades NETs and induces apoptosis 5 . Notably, colicin E9 from Escherichia coli is the first identified bacterial PFT with nuclease activity, enabling DNA damage with the host cytoplasm 6,7 . Thermostable direct hemolysin (TDH) is a PFT and a major virulence factor of pathogenic Vibrio species, including V. parahaemolyticus (Vp-TDH), V. cholera non-O1, V. mimicus, V. alginolyticus, and Grimontia hollisae (formally described as V. hollisae ) etc 8–13 . Ingesting raw seafood or exposure to wounds contaminated with Vibrio TDH can lead to hypovolemic shock, bacteremia, and sepsis 14–22 . Studies have shown that TDH from G. hollisae (Gh-TDH) induces osmotic lysis of red blood cells 23,24 , and exhibits cyto-, cardio-, entero-, and hepato-toxicity in cultured cells, as well as lethality in mice 16,24–30 . Interestingly, while Gh-TDH binds erythrocyte membranes across species with similar affinity, its hemolytic activity varies, suggesting additional activities or factors contribute to its pathogenicity 31 . Additionally, an "Arrhenius effect" has been observed, where Gh-TDH is detoxified at 60–70 o C but reactivates above 80 o C 24,32 , implicating multiple promoter interfaces in its oligomerization. Crystal structure analysis reveals three distinct tetrameric oligomeric conformations of Gh-TDH: oligomer I, similar to Vp-TDH, and oligomers II and III, which share a dimer motif. This dimer likely represents the minimal structural unit required for membrane binding and hemolytic activity, highlighting the structural complexity of Gh-TDH's role in pathogenesis 30 . Incubation of fluorescent recombinant Gh-TDH-FITC with liver cells demonstrated that Gh-TDH binds to the cell membrane, induces extensive membrane blebbing, translocates to the nucleus, and triggers cell lysis and death 16 . This cytotoxic mechanism shares similarities with glucocorticoids and toxic compounds, which induce apoptosis through activation of intracellular receptors that translocate to the nucleus, regulate gene expression, and activate endonucleases 33 . These findings suggest that Gh-TDH may enhance its virulence by either activating endogenous nuclease-dependent apoptosis pathways or directly cleaving DNA through intrinsic nuclease activity, thereby amplifying its cytotoxic potential 34–36 . This study investigated whether Gh-TDH directly degrades cellular DNA or activates endogenous nucleases. In vitro nuclease activity assays and X-ray crystallography revealed the structural and mechanistic basis of Gh-TDH's nuclease activity. The assays demonstrated that Gh-TDH binds DNA and catalyzes 3'–5' exonucleolysis, producing a 3-mer product. Structure analysis of the Gh-TDH-DNA complexs revealed a unique DNA-binding and cleavage mechanism, as Gh-TDH lacks the canonical catalytic motifs typical of 3'–5' exonucleases, underscoring its distinct nuclease properties 37 . Results Characterization of 3'–5' exonuclease activity of Gh-TDH Recombinant wild-type and mutant Gh-TDH proteins were expressed and produced in E. coli BL21(DE3)pLysS and purified for biochemical and structural studies. To evaluate nuclease activity and substrate specificity, various DNA and RNA substrates, including 5'- or 3'-FAM or biotin-labeled single- and double-stranded DNAs, stem-loop dsDNA or dsRNA with varying 5'- or 3'-overhang lengths, and poly(dA 12 ), poly(dT 12 ), and poly(dC 12 ), were synthesized (Supplementary Table 1). Gh-TDH efficiently digested 5'-FAM-labeled stem-loop DNA in a concentration-dependent manner, producing a 3-nt 5'-FAM-labeled fragment through 3'–5' exonucleolysis (Fig. 1 a, lanes 1–5 and 11–15). However, activity on 3'-FAM-labeled stem-loop substrates was significantly reduced, with cleavage of the FAM tag observed only at high enzyme concentrations (Fig. 1 a, lanes 6–10 and 16–20). When incubated with 5'-FAM-labeled stem-loop dsDNA containing varying 3'-overhang lengths, Gh-TDH digested the overhangs to form blunt-ended dsDNA intermediates (Fig. 1 b). These results indicate that Gh-TDH preferentially trims single-stranded 3'-overhangs in a 3'–5' exonucleolytic manner before processing dsDNA stem-loops. The 3'-FAM tag inhibits nuclease activity, reducing cleavage efficiency (Fig. 1 a). Regardless of overhang length, Gh-TDH processes DNA until a blunt-ended dsDNA structure is formed (Fig. 1 b), highlighting its substrate preference for 3'-overhangs. Gh-TDH's ribonuclease activity was evaluated using 5'-FAM-labeled stem-loop dsRNA with 4-nt 3'-overhang. The enzyme cleaved RNA but exhibited ~ 70% lower efficiency compared to its deoxyribonuclease activity (Supplementary Fig. 1). Substrate specificity studies using 5'-FAM-labeled poly(dA 12 ), poly(dT 12 ), and poly(dC 12 ) showed that Gh-TDH efficiently degraded poly(dA 12 ) and poly(dT 12 ) in a time-dependent manner, with significantly reduced activity on poly(dC 12 ). The cleavage produced final 3-nt fragments, resulting in poly(dA 3 ), poly(dT 3 ), and poly(dC 3 ) products (Supplementary Fig. 2). The influence of divalent metal ions on Gh-TDH's 3'–5' exonuclease activity was also investigated. Mg 2+ and Co 2+ significantly enhanced activity, Mn 2+ exerted a marginal effect, while Ca 2+ , Ni 2+ , Cu 2+ , and Zn 2+ showed no enhancement (Supplementary Fig. 3a). Analysis of Mg 2+ concentrations showed that Gh-TDH exhibited optimal activity at 5–10 mM, while higher concentrations leading to a dose-dependent decline (Supplementary Fig. 3b). In addition, ethylenediaminetetraacetic acid (EDTA) inhibited nuclease activity, confirming the requirement of divalent metal ions for Gh-TDH function (Supplementary Fig. 3b). Additionally, the final DNA cleavage product matched the size of the synthetic 3-mer sequence CTA (Supplementary Fig. 3c). Overall structure of Gh-TDH-DNA complexes To investigate how Gh-TDH processes DNA, we determined its crystal structure bound to a 12-nt ssDNA using molecular replacement with the Gh-TDH structure (PDB entry: 4WX3) 30 . Crystallization conditions, data collection, and refinement statistics are detailed in Supplementary Tables 2 and 3. Two Gh-TDH-DNA complexes (complexes I and II), differing in N -terminal conformation, were obtained under distinct crystallization conditions, both with identical lattice packing and space group (P42 1 2). In both complexes, eight ssDNA strands form four duplexes, each comprising 10 base-paired (C 3 –G 12 ) nucleotides and a 2-nt unpaired 5'-overhang. One end of the duplexes binds the same Gh-TDH tetramer, while the other end associates with protomers from four different tetramers (Fig. 2 a,b). Complex I showed disordered N -terminal residues 1–11 (Fig. 2 a,c), while in complex II, residues 7–11 are ordered and inserted into the tetramer's central pore (Fig. 2 b,c). Structure superposition reveals a conformational shift starting at Asp14 (D14), with a β-sheet stabilized by Gly62 (G62) and D14 in complex I replaced by a loop in complex II (Fig. 2 c). Despite N -terminal differences, the DNA conformation remains unchanged, suggesting N -terminal region variability do not affect DNA binding. Both complexes exhibited a narrowed central pore (19 Å vs. 20 Å in Gh-TDH) and a slight structural adjustment, with complex I showing a 5 o rotation along the x-axis, resulting in a root mean square deviation (r.m.s.d.) of 1.54 Å relative to the Gh-TDH structure (Supplementary Fig. 4). These findings indicate minor conformational changes in the Gh-TDH tetramer accommodate binding of four duplexes. The crystal structures of complexes I and II revealed that Tyr87 (Y87) of the Gh-TDH protomer caps the 5'-end of each DNA duplex (Fig. 3 ). In Gh-TDH, residues Y87–G90 form a β-hairpin structure between the sixth and seventh β-strands (Fig. 3 a). Upon DNA binding, these residues undergo conformational changes to interact with the C 3 -G 12 base pair (C 3 on the sense strand and G 12 on the anti-sense strand) (Fig. 3 b and Supplementary Fig. 4b). Key interactions include the indole side chain of Trp65 (W65), which forms a 2.8 Å hydrogen bond with the carbonyl group of C 3 , and the phenolic ring of Y87, which engages in π-π stacking (3.9 Å) with C 3 's aromatic pyrimidine ring. In the β-turn, the main chain amino group of Asp89 (D89) hydrogen bonds with the N7 amine (2.8 Å) and carbonyl group (3.4 Å) of G 12 , while the amino group of Gly90 (G90) forms a 3.0 Å hydrogen bond with G 12 's carbonyl group. Additionally, the side chains of Lys88 (K88) and D89 create water-mediated hydrogen bonds with the phosphate backbone between G 11 and G 12 . In complex I, interactions between K88, D89, and the 3'-end of the dsDNA stabilize their structural flexibility. This stabilization is reflected in the reduced B-factors for K88's amino group in Gh-TDH compared to that in complex I (52.9 to 21.0). The elasticity of residues Y87–G90's β-hairpin structure facilitates Gh-TDH's capacity to adopt and bind diverse DNA structures effectively. Gh-TDH recognizes different dsDNA substrates in a similar mode To evaluate the impact of DNA sequence and length on Gh-TDH's binding and cleavage activity, 10-nt and 6-nt DNA substrates (with or without 5'-FAM-labels) were used in nuclease assays and structural analysis (complexes III and IV). As shown in Fig. 4 a, Gh-TDH cleaved both 10-nt and 6-nt DNAs in a concentration-dependent manner. Cleavage sites were identified through high-resolution mass analysis (HD Q-TOF) of a 6-nt DNA substrate with hydroxyl groups at both ends (Fig. 4 b). The analysis revealed a molecular mass of 845.2002, matching the calculated mass of a CTA 3-mer with a 3'-hydroxyl group (Mw calc .: 845.2015) (Fig. 4 c), confirming that Gh-TDH cleaves 3' O-P bond to produce a 3-mer DNA with 3'-hydroxyl group. Structural analysis of complexes III and IV revealed that the 10-nt and 6-nt ssDNA substrates used for co-crystallization are self-complemented, forming blunt-ended dsDNA (Fig. 5 a,b). The N -terminal residues Pro7–Pro11, visible in complexes II and III, were absent in complex IV, where the N -terminus remained disordered, resembling complex I and the Gh-TDH structure (Fig. 2 c and Fig. 5 a,b). Despite differences in DNA length and sequence (A 9 and A 5 in complexes III and IV, vs. G 11 in complex I) (Supplementary Table 2), the binding modes in complexes III and IV closely resemble that of complex I (Fig. 5 a,b). In complex III, the indole side chain of W65 forms a hydrogen bond with C 1 's carbonyl group (2.9 Å, sense strand), Y87's phenolic side chain stacks with C 1 (3.7 Å), and D89's main chain amino group forms hydrogen bonds with G 10 's (antisense strand) N7 amine (2.8 Å) and carbonyl group (3.5 Å). G90's main chain amino group forms hydrogen bonds with A 9 's N7 amine (3.5 Å) and G 10 's carbonyl group (2.9 Å) in complex III (Fig. 5 a), compared to a hydrogen bond with only the G 12 's carbonyl group (2.9 Å) in complex I (Fig. 2 b). Complex IV shows a nearly identical DNA-binding environment to complex III (Fig. 5 b), with consistent interaction regions across the Gh-TDH-DNA complexes (Fig. 5 c). In addition, variations in length result only in a shift of the DNA helix, as evidenced by the 45° rotation of the helix between complex I and complex IV (Fig. 5 d). This result suggests that Gh-TDH binds to dsDNA similarly, regardless of DNA length and sequence. Lys88 is an important residue for DNA cleavage Alanine scanning mutagenesis of Gh-TDH residues near the DNA-binding site (W65, Y87, K88, D89, Y107A, and Y87/Y107) was performed to assess their role in nuclease activity using a 5'-FAM-labeled stem-loop dsDNA with a 4-nt 3'-overhang. The K88A variant showed the most significant reduction, completely losing exonuclease activity at 5 µM protein concentration (Fig. 6 a,b). Mutations of Y87A, D89A, and Y107A had minimal impact, while W65A and Y87A/Y107A caused partial reductions. Further testing of K88 mutants (K88E, K88D, K88Q, K88N, K88R, and K88H) showed that K88E, K88D, K88Q, and K88N reduced activity to 66%, 49%, 49%, and 54%, respectively (Fig. 6 a,c), while K88R retained wild-type activity and K88H showed a slight increase. These results suggest that a positively charged residue at position 88 is important for DNA cleavage. Dissociation constants ( K D ) for wild-type Gh-TDH and the K88A variant, measured by bio-layer interferometry (BLI), were 14.8 and 22.4 µM, respectively, indicating that the loss of exonuclease activity in the K88A variant is not attributable to changes in substrate binding affinity (Supplementary Fig. 5). To investigate the effect of K88 mutation on DNA binding and trimming, wild-type Gh-TDH and the K88A variant were co-crystallized with an 11-nt ssDNA, forming complex V and complex VI, respectively (Supplementary Tables 2 and 3). In solution, the two ssDNA strands form a dsDNA with a 1-nt 3'-overhang and a 10-nt duplex. In both complexes, the first 11 N -terminal residues are disordered, as seen in Gh-TDH and complex I (Fig. 5 e,f). Crystal structures revealed that A 11 of the antisense strand forms sugar-π and π-π interactions with W65 and Y107, respectively, differing from the C 3 /G 12 , C 1 /G 10 , and C 1 /G 6 base pairs observed in complexes I-IV (Figs. 3 b and 5 ). In addition, Y87 still forms π-π stacking with C 1 , but C 1 shifts closer to the hydroxyl group of Y87 (Fig. 5 g). In complexes V and VI, D89 forms a hydrogen bond with the N7 amine of G 10 (the penultimate base), similar to its interaction with the final G base in complexes I-IV. However, in complexes V and VI, G90 forms a hydrogen bond with the N7 amine of G 10 (the penultimate G), unlike in complexes I-IV, where G90 forms a hydrogen bond with the carbonyl group of the final G (G 12 in complexes I and II, G 10 in complex III, G 6 in complex IV) (Figs. 3 b and 5 ). Additionally, G90's carbonyl group forms a hydrogen bond with the C-4 amino group of C 1 (sense strand) in complexes V and VI, a bond absent in complexes I–IV. Water molecules bound to K88 and D89 in complexes I-III are not observed in complexes IV-VI (Supplementary Fig. 6a–f). Structural analysis of complexes V and VI, along with BLI results, supports that the K88A variant does not affect Gh-TDH's binding affinity or mode, reinforcing K88's role as a catalytic residue. The differences in binding patterns between complexes III and V suggest that Gh-TDH adopts to various DNA structures to facilitate cleavage. (Fig. 5 g). Discussion This study is the first to identify Gh-TDH, a hemolysin in Vibrio species, with nuclease activity, revealing its distinct structural basis compared to other known exonucleases. Gh-TDH shows no significant sequence or structural similarity to known nucleases. Unlike well-characterized 3'–5' exonucleases or other nucleases (including the DEDD and DEK superfamilies), which typically use aspartate (Asp) or glutamate (Glu) residues to coordinate divalent metal ions and stabilize the transition state during phosphodiester bond cleavage 37,38 , Gh-TDH primarily relies on aromatic residues Trp65 and Tyr87 to interact with the non-scissile strand. Structural analysis shows that members of the DEDD superfamily, such as ExoX and TREX2, utilize a loop between β1 and β2 strands or α-helical loop structure to specifically bind to the non-scissile strand of dsDNA 39,40 . Additionally, while the general base in DEDD family enzymes is typically located within a loop (e.g., TREX2's α6–α7 loop) 37 , in Gh-TDH, the general base Lys88 is located within a unique β-turn (K88 to G90) which plays a critical role in DNA binding and cleavage. Gh-TDH is a metal ion-dependent nuclease that lacks the typical conserved motif of 3'–5' exonucleases. Mutational analysis of acidic residues near the DNA-binding site (D89, D117, E118) showed no significant effect on nuclease activity (Supplementary Fig. 6g), indicating these residues may play supporting roles in coordinating the divalent metal ion. While most metal-dependent nucleases use a bimetallic ion mechanism, monometallic ion mechanisms are more common in endonucleases 37 . Notably, in the bimetallic ion nuclease Ape1 (PDB entry: 7CD5), the divalent metal ions were not determined in the crystal structure and was proposed to be captured in its product-bound form 41 . The metal ion dependency of Gh-TDH, coupled with the absence of detectable divalent ions in the Gh-TDH-DNA complex, suggests the complex may also be in a product-bound state. In most nucleases, histidine or tyrosine residue near the active site usually functions as a general base, activating water molecules to initiate DNA cleavage 37 . However, mutations of Y87 and Y107 in Gh-TDH had little effect on activity (Fig. 6 ). In contrast, mutations at K88 significantly affected activity. Structural analysis revealed that K88 interacts with the DNA 3'-end through a water-mediated network involving water molecules W 2 , W 3 , and W 4 (Supplementary Fig. 6a–c). This function is analogous to the role of K131 in the bacteriophage lambda exonuclease as a general base 42 . K88 in Gh-TDH likely plays a central role as a general base for activating water molecules during the catalytic process. Structural comparison of complexes I–VI revealed only minor conformational changes in the hydrophobic pocket residues Y87, W65, and Y107, despite variations in DNA binding. A consistent π-π stacking interaction between Y87 and the first cytosine (sense strand) was observed across all complexes, with slight conformational shifts in complexes V and VI (Fig. 5 g). In complexes I–IV, W65 forms a hydrogen bond with the first cytosine (sense strand), while in complexes V and VI, a sugar-π interaction with A 11 (antisense strand) is observed (Fig. 5 ). Y107 interacts minimally with DNA in complexes I–IV, but forms a π-π interaction with A 11 (antisense strand) in complexes V and VI. These shifts likely enable Gh-TDH to distinguish between scissile and non-scissile strands, facilitating precise cleavage at the 3'-end of the ssDNA substrate (Fig. 5 g). The reduced exonuclease activity of the W65A and Y87A/Y107A variants further highlights the importance of these residues in efficient DNA cleavage (Fig. 6 ). Similar aromatic residues in RNase T (Phe29) and TREX2 (Tyr129), members of the DEDD 3'–5' exonuclease superfamily, also play key roles in precise 3'-overhang cleavage 39,40,43–45 . The Gh-TDH-DNA complexes revealed the structure of five additional amino acids ( 7 PFPAP 11 ) at the N -terminus, a region critical for hemolytic activity and pore formation in Gh-TDH and Vp-TDH 30,46,47 . Despite its importance, the mechanism of pore formation remains unclear, as the N -terminal region is disordered in both TDH crystal structures. Cryo-EM analysis of Vp-TDH shows NTR density maps associated with pore-forming structures, but its exact role in pore formation is yet to be determined 48 . The complex structures determined in this study provide evidence that the N -terminal region undergoes two distinct conformational changes during the pore-forming process. Our findings highlight the presence of these additional amino acids ( 7 PFPAP 11 ) in complexes II and III, which may provide valuable insights into the functional significance of NTR. Conclusion Nucleases are vital for DNA repair and genome integrity, with human nucleases like TREX1 and TREX2 maintaining stability 40,45,49 . In contrast, pathogenic bacterial nucleases, such as Cdt, can enter the host nucleus, targeting host DNA and disrupting host nucleases, leading to DNA damage and cell death 50 . This study identifies Gh-TDH as the first Vibrio hemolysin with nuclease activity. Through in vitro assays, mass spectrometry, structural analysis, and mutagenesis, we confirm Gh-TDH as a novel 3'–5' exonuclease with both hemolytic and nuclease functions. These findings lay the groundwork for targeted therapeutic strategies against Vibrio infections, with future research focusing on Gh-TDH's membrane interactions and permeabilization. Materials and Methods Molecular cloning, mutagenesis, gene expression, protein production and purification of the recombinant Gh-TDH wild-type and the mutant variants The Gh-TDH gene (GenBank accession ID: WP_040528653.1) was cloned into the pCR2.1-TOPO vector. For the mutant variants, the plasmids were constructed by the QuikChange site-directed mutagenesis method. All plasmids were confirmed via DNA sequencing. Each of the wild-type and the mutant constructs was transformed into E. coli BL21(DE3)pLysS cells for gene expression. Gene expression, protein production, and purification were carried out using previously established methods, yielding highly purified proteins for biochemical and structural analysis 24,29 . The protein purity was assessed by SDS-PAGE. All wild-type and the mutant variants of Gh-TDH proteins were concentrated using Amicon Ultra-15 3,000 NMWL concentrator (Merck) in 20 mM Tris-HCl at pH 7.0 for the following nuclease activity assay, BLI analysis, and protein crystallization. Nuclease activity assay Fluorescein amidite (FAM)-labeled DNA substrates (3'- or 5'-end) were synthesized by Genomics Inc. Taiwan for nuclease activity assays. The sequences of DNA substrates used in nuclease activity are listed in Supplementary Table 1. In nuclease activity assays, 0.5 µM DNA substrates were incubated with protein in 5 mM MgCl 2 and 20 mM Tris-HCl (pH 7.0). After incubation at 37 ℃ for 60 min, 2x TBE-urea loading buffer (G-Biosciences) was added to quench the traction at 95 ℃ for 5 min. DNA digestion patterns were analyzed using 20% TBE urea polyacrylamide gels and visualized under blue light. Nuclease-specific activity was measured using 5 µM protein with 0.5 µM substrate, with excision quantified using ImageJ. Crystallization, data collection, and structure determination The purified Gh-TDH wild-type, and K88A variant were mixed with ssDNA at a 1:1.3 molar ratio, respectively, and incubated on ice for 20 minutes. The mixtures were crystallized using the hanging-drop vapor-diffusion method at 20°C. The crystallization conditions and the sequences of co-crystallized ssDNA for complexes I–VI were listed in Supplementary Table 2. Crystals were cryo-protected with Paraton-N (Hampton Research) for data collection at BL15-A1, TPS-05A, and TPS-07A (NSRRC, Taiwan, R.O.C.). All diffraction data were indexed and scaled with HKL2000 51 . Molecular replacement was performed using Gh-TDH (PDB entry: 4WX3) as the search model 30 in MOLREP of CCP4 52 . Models were built in COOT 53 and refined with REFMAC 54 . The atomic coordinates and structure factors of complexes I–VI have been deposited in the Protein Data Bank with the accession code 9LCS, 9L8E, 9L9M, 9LCM, 9LCU, and 9LCW, respectively. Data processing and refinement statistics are summarized in Supplementary Table 3. Mass spectrometric analysis of Gh-TDH DNA cleavage products Mass spectrometric analyses were performed by the Center for Advanced Instrumentation and Department of Applied Chemistry at National Yang Ming Chiao Tung University, Hsinchu, Taiwan, R.O.C. To analyze the molecular mass of DNA products cleaved by Gh-TDH, a 6-mer DNA substrate (CTATAG) (50 µM) was incubated with Gh-TDH (50 µM) in 20 mM Tris-HCl (pH 7.0) and 5 mM MgCl 2 at 37°C for 4 hours. To quench the reaction, the mixture was heated to 60°C for 10 min. The reaction mixture was centrifuged at 13,000 rpm to remove the fibrillar Gh-TDH and the supernatant was subjected to HD Q-TOF with ESI(+)MS analysis (Bruker). The collected data were analyzed using Compass DataAnalysis 4.1 (Bruker). Bio-layer interferometry (BLI) binding kinetics assay The binding affinity of 5'-biotinylated stem-loop dsDNA to Gh-TDH wild-type or the K88A variant was measured using the Octet HTX system (FroteBio) at the Center for Emergent Functional Matter Science, National Yang Ming Chiao Tung University, Taiwan, R.O.C. BLI measurements were carried out at a shaking speed of 1,000 rpm. The purified Gh-TDH proteins at various concentrations of 10, 20, 30, 40, and 50 µM were prepared in kinetic buffer (PBS at pH 7.4, 50 mM EDTA, 1 mg/mL BSA) and loaded onto HIS1K (streptavidin) biosensors (Molecular Devices, ForteBio). Five concentrations of Gh-TDH in kinetic buffer were added to a black polypropylene 96-well microplate (Greiner Bio-one), with one row containing kinetic buffer as a reference control. Each protein concentration underwent an assay cycle using streptavidin-5'-biotinylated stem-loop dsDNA probes and blank probes. One assay cycle consists of 60 s of baseline normalization in kinetics buffer, 400 s of association in the protein solution, 200 s of dissociation in kinetics buffer, and 30 s of regeneration in 0.5 M NaOH. BLI results were analyzed using FortéBio Data Analysis High Throughput 12.0. The sequences of ssDNA substrates used in BLI assays are listed in Supplementary Table 1. Declarations Data Availability The data associated with this study are available within the article and Supplementary Information. All the coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 9LCS, 9L8E, 9L9M, 9LCM, 9LCU, and 9LCW. Source data are provided with this paper. All other data are available from the corresponding author on request. Acknowledgements This work was supported by the National Science and Technology Council (NSTC), Taiwan (grants 110-2113-M-A49-026-MY3, 113-2113-M-A49-025-, and 113-2113-M-A49-018-). This work is also supported in part by the Kaohsiung Medical University Research Center Grant (NYCUKMU-114-I005) and the Center for Emergent Functional Matter Science and the Center for Intelligent Drug Systems and Smart Biodevices (IDS 2 B) of National Yang Ming Chiao Tung University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. The authors thank the experimental facility and the technical service provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology and the National Synchrotron Radiation Research Center (NSRRC), a national user facility supported by NSTC, Taiwan. Author Contributions P.Y.H., T.K.W., and C.Y.C. conceived the project, designed the experiments, and wrote the manuscript; P.Y.H., Y.K.W., and S.C.H. performed molecular cloning, protein production, and protein purification; P.Y.H., F.P.C., and T.Y.H. performed binding kinetics and BLI analysis; P.Y.H., Y.C.L., and Y.M.K. performed protein crystallization, diffraction data collection, and structural determination; P.Y.H. performed nuclease activity assay and mass spectrometric analysis. Competing Interests The authors declare no conflict of interest. References Mondal, A. K. et al. Glu289 residue in the pore-forming motif of Vibrio cholerae cytolysin is important for efficient beta-barrel pore formation. J Biol Chem 298 , 102441 (2022). Chiu, Y. C. et al. Structural basis for calcium-stimulating pore formation of Vibrio alpha-hemolysin. Nat Commun 14 , 5946 (2023). Beiter, K. et al. An endonuclease allows Streptococcus pneumoniae to escape from neutrophil extracellular traps. 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Purification and partial characterization of a Non-01 Vibrio cholerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaemolyticus . Infection and Immunity 52 , 319-322 (1986). Shinoda, S. et al. Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins. Microbiol Immunol 48 , 547-551 (2004). Gonzalez-Escalona, N., Blackstone, G. M. & DePaola, A. Characterization of a Vibrio alginolyticus strain, isolated from Alaskan oysters, carrying a hemolysin gene similar to the thermostable direct hemolysin-related hemolysin gene ( trh ) of Vibrio parahaemolyticus . Appl Environ Microbiol 72 , 7925-7929 (2006). Yoh, M., Honda, T. & Miwatani, T. Purification and partial characterization of a Vibrio hollisae hemolysin that relates to the thermostable direct hemolysin of Vibrio parahaemolyticus . Can J Microbiol 32 , 632-636 (1986). Nishibuchi, M., Janda, J. M. & Ezaki, T. The thermostable direct hemolysin gene ( tdh ) of Vibrio hollisae is dissimilar in prevalence to and phylogenetically distant from the tdh genes of other vibrios : implications in the horizontal transfer of the tdh gene. Microbiol Immunol 40 , 59-65 (1996). Goshima, K., Honda, T., Hirata, M., Kikuchi, K. & Takeda, Y. Stopping of the spontaneous beating of mouse and rat myocardial cells in vitro by a toxin from Vibrio parahaemolyticus . J Mol Cell Cardiol 9 , 191-213 (1977). Lang, P. A. et al. Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes. Cell Microbiol 6 , 391-400 (2004). Lin, Y. R. et al. The thermostable direct hemolysin from Grimontia hollisae causes acute hepatotoxicity in vitro and in vivo . PLoS One 8 , e56226 (2013). Miyamoto, Y. et al. In vitro hemolytic characteristic of Vibrio parahaemolyticus : its close correlation with human pathogenicity. J Bacteriol 100 , 1147-1149 (1969). Naim, R., Yanagihara, I., Iida, T. & Honda, T. Vibrio parahaemolyticus thermostable direct hemolysin can induce an apoptotic cell death in Rat-1 cells from inside and ouside of the cells. Fems Microbiol Lett 195 , 237-244 (2001). Raimondi, F. et al. Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin in in vitro systems. Infect Immun 68 , 3180-3185 (2000). Sakurai, J., Honda, T., Jinguji, Y., Arita, M. & Miwatani, T. Cytotoxic effect of the thermostable direct hemolysin produced by Vibrio parahaemolyticus on FL cells. Infect Immun 13 , 876-883 (1976). Tang, G. Q., Iida, T., Yamamoto, K. & Honda, T. Ca 2+ -independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on Intestine-407, a cell-line derived from human embryonic intestine. Fems Microbiol Lett 134 , 233-238 (1995). Cai, Q. & Zhang, Y. Structure, function and regulation of the thermostable direct hemolysin (TDH) in pandemic Vibrio parahaemolyticus . Microb Pathog 123 , 242-245 (2018). Fabbri, A. et al. Vibrio parahaemolyticus thermostable direct hemolysin modulates cytoskeletal organization and calcium homeostasis in intestinal cultured cells. Infect Immun 67 , 1139-1148 (1999). Wang, Y. K. et al. Site-directed mutations of thermostable direct hemolysin from Grimontia hollisae alter its Arrhenius effect and biophysical properties. International Journal of Biological Sciences 7 , 333-346 (2011). Hiyoshi, H., Kodama, T., Iida, T. & Honda, T. Contribution of Vibrio parahaemolyticus virulence factors to cytotoxicity, enterotoxicity, and lethality in mice. Infect Immun 78 , 1772-1780 (2010). Honda, T., Goshima, K., Takeda, Y., Sugino, Y. & Miwatani, T. Demonstration of the cardiotoxicity of the thermostable direct hemolysin (lethal toxin) produced by Vibrio parahaemolyticus . Infect Immun 13 , 163-171 (1976). Miyamoto, S. & Kuroda, K. Lethal effect of fresh sea water on Vibrio parahaemolyticus and isolation of Bdellovibrio parasitic against the organism. Jpn J Microbiol 19 , 309-317 (1975). Huang, S. C. et al. Potential antitumor therapeutic application of Grimontia hollisae thermostable direct hemolysin mutants. Cancer Sci 106 , 447-454 (2015). Wang, Y. K. et al. Purification, crystallization and preliminary X-ray analysis of a thermostable direct haemolysin from Grimontia hollisae . Acta Crystallogr Sect F Struct Biol Cryst Commun 67 , 224-227 (2011). Wang, Y. K. et al. Multiple pleomorphic tetramers of thermostable direct hemolysin from Grimontia hollisae in exerting hemolysis and membrane binding. Sci Rep 9 , 9833 (2019). Yoh, M., Morinaga, N., Noda, M. & Honda, T. The binding of Vibrio parahaemolyticus 125 I-labeled thermostable directhemolysin to erythrocytes. Toxicon 33 , 651-657 (1995). Ohnishi, K. et al. Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus . Fems Microbiol Lett 318 , 10-17 (2011). Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284 , 555-556 (1980). Peitsch, M. C. et al. Characterization of the endogenous deoxyribonuclease involved in nuclear DNA degradation during apoptosis (programmed cell death). EMBO J 12 , 371-377 (1993). Samejima, K. & Earnshaw, W. C. Trashing the genome: the role of nucleases during apoptosis. Nat Rev Mol Cell Biol 6 , 677-688 (2005). Berends, E. T. et al. Nuclease expression by Staphylococcus aureus facilitates escape from neutrophil extracellular traps. J Innate Immun 2 , 576-586 (2010). Yang, W. Nucleases: diversity of structure, function and mechanism. Q Rev Biophys 44 , 1-93 (2011). Singleton, M. R., Dillingham, M. S., Gaudier, M., Kowalczykowski, S. C. & Wigley, D. B. Crystal structure of RecBCD enzyme reveals a machine for processing DNA breaks. Nature 432 , 187-193 (2004). Wang, T. et al. Recognition and processing of double-stranded DNA by ExoX, a distributive 3'–5' exonuclease. Nucleic Acids Res 41 , 7556-7565 (2013). Cheng, H. L. et al. Structural insights into the duplex DNA processing of TREX2. Nucleic Acids Research 46 , 12166-12176 (2018). Liu, T. C. et al. APE1 distinguishes DNA substrates in exonucleolytic cleavage by induced space-filling. Nat Commun 12 , 601 (2021). Zhang, J., Pan, X. & Bell, C. E. Crystal structure of lambda exonuclease in complex with DNA and Ca 2+ . Biochemistry 53 , 7415-7425 (2014). Brucet, M. et al. Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains. J Biol Chem 282 , 14547-14557 (2007). Hsiao, Y. Y. et al. Structural basis for RNA trimming by RNase T in stable RNA 3'-end maturation. Nat Chem Biol 7 , 236-243 (2011). Zhou, W., Richmond-Buccola, D., Wang, Q. & Kranzusch, P. J. Structural basis of human TREX1 DNA degradation and autoimmune disease. Nat Commun 13 , 4277 (2022). Kundu, N., Tichkule, S., Pandit, S. B. & Chattopadhyay, K. Disulphide bond restrains the C-terminal region of thermostable direct hemolysin during folding to promote oligomerization. Biochem J 474 , 317-331 (2017). Verma, P. & Chattopadhyay, K. Current perspective on the membrane-damaging action of thermostable direct hemolysin, an atypical bacterial pore-forming toxin. Front Mol Biosci 8 , 717147 (2021). Mishra, S. et al. Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM. Proteins 91 , 137-146 (2023). Wang, Q., Du, J., Hua, S. C. & Zhao, K. TREX1 plays multiple roles in human diseases. Cell Immunol 375 (2022). Nesic, D., Hsu, Y. & Stebbins, C. E. Assembly and function of a bacterial genotoxin. Nature 429 , 429-433 (2004). Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276 , 307-326 (1997). Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr 59 , 1131-1137 (2003). Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60 , 2126-2132 (2004). Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53 , 240-255 (1997). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryinformation.docx structures.zip 1 (crystal structure data) Cite Share Download PDF Status: Under Review Version 1 posted 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. 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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-5915599","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":415419841,"identity":"fe4d16ef-d6f8-4cf0-8779-b5d8c2c6a243","order_by":0,"name":"Chin-Yuan 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University","correspondingAuthor":false,"prefix":"","firstName":"Tung-Kung","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-01-28 01:55:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5915599/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5915599/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77398211,"identity":"bbe6ed5e-dfd8-4115-9ae6-42a6cbcc94b5","added_by":"auto","created_at":"2025-02-28 08:03:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":186158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNuclease activity assay of Gh-TDH.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Gh-TDH was incubated with 0.5 mM FAM-labeled stem-loop dsDNA substrates, including 5'-FAM-labeled 4-nt 3'-overhang stem-loop dsDNA (Lane 1–5), 3'-FAM-labeled 4-nt 3'-overhang stem-loop dsDNA (Lane 6–10), 5'-FAM-labeled 4-nt 5'-overhang stem-loop dsDNA (Lane 11–15), and 3'-FAM-labeled 4-nt 5'-overhang stem-loop dsDNA (Lane 16–20). \u003cstrong\u003eb, \u003c/strong\u003eGh-TDH was incubated with 0.5 mM 5'-FAM-labeled stem-loop dsDNA with varying 3'-end protrusions: blunt-ended dsDNA (Lane 1–5), 2-nt 3'-overhang (Lane 6–10), 4-nt 3'-overhang (Lane 11–15), and 8-nt 3'-overhang (Lane 16–20).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/1f6147b507c97614e2feeaa0.png"},{"id":77397759,"identity":"68712b1a-aa54-4036-ab0d-b3c5961a29f5","added_by":"auto","created_at":"2025-02-28 07:55:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1401762,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal structures of Gh-TDH-DNA complexes I and II.\u003c/strong\u003e \u003cstrong\u003ea, \u003c/strong\u003eSide (left) and top (right) views of complex I (right). \u003cstrong\u003eb,\u003c/strong\u003e Side (left) and top (right) views of complex II (right). The \u003cem\u003eN\u003c/em\u003e-terminal region (NTR) is framed by a dashed box. \u003cstrong\u003ec,\u003c/strong\u003e Comparison of \u003cem\u003eN\u003c/em\u003e-terminal conformations in Gh-TDH, complex I, and complex II, with residues Pro7–Gly12 shown in dark purple and G62 in cyan.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/8ebfe70609d3c0f6e39b83ab.png"},{"id":77397762,"identity":"cf74a30b-e334-401a-a660-7a41f1c18556","added_by":"auto","created_at":"2025-02-28 07:55:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":497309,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural analyses of the dsDNA-binding site in the Gh-TDH-DNA complex.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e A protomer of tetrameric Gh-TDH is shown in blue, with the DNA-binding sites highlighted as balls and sticks. \u003cstrong\u003eb, \u003c/strong\u003eA local view of protein-DNA interactions at the DNA terminal, where the sense strand is shown in green and the antisense strand in white. The residues involved in DNA binding are depicted in balls and sticks. The water molecule is shown as red sphere (W\u003csub\u003e1\u003c/sub\u003e). Hydrogen bond interactions between the C\u003csub\u003e3\u003c/sub\u003e-G\u003csub\u003e12\u003c/sub\u003e pair of the dsDNA and the b-hairpin (\u003csup\u003e87\u003c/sup\u003eYKDG\u003csup\u003e90\u003c/sup\u003e) of the protein are shown in yellow dashed lines.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/872e09eff7d472ae9daa67d0.png"},{"id":77397760,"identity":"8ffe5392-46d9-418b-a59e-0b2c874fc4cd","added_by":"auto","created_at":"2025-02-28 07:55:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":281588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNuclease activity assay of Gh-TDH against 6- and 10-nt DNA substrates.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Exonuclease activity of Gh-TDH digested 5'-FAM labeled 6-nt and 10-nt ssDNA. \u003cstrong\u003eb,\u003c/strong\u003e HD Q-TOF spectrum of 6-nt 5'-OH-CTATAG-OH-3' used for Gh-TDH nuclease activity assay. \u003cstrong\u003ec,\u003c/strong\u003e HD Q-TOF spectrum of Gh-TDH cleaved 6-nt 5'-OH-CTATAG-OH-3'. The singly charged ion 5'-OH-CTA-OH-3' at m/z 845.2002 was observed in the Gh-TDH DNA cleavage product. \u003cstrong\u003ed,\u003c/strong\u003e Chemical structure of 6-nt 5'-OH-CTATAG-OH-3' and the site of Gh-TDH cleavage to 3-nt 5'-OH-CTA-OH-3' shown in bracket.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/d25bb9bc7977f5af188176ef.png"},{"id":77397764,"identity":"1f568401-4669-4090-bc07-6814b56a038a","added_by":"auto","created_at":"2025-02-28 07:55:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":841254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of Gh-TDH to DNA ends with different sequences.\u003c/strong\u003e \u003cstrong\u003ea,\u003c/strong\u003e Complex III:\u003cstrong\u003e \u003c/strong\u003ecrystal structure of Gh-TDH in complex with 10-nt dsDNA. \u003cstrong\u003eb, \u003c/strong\u003eComplex IV: crystal structure of Gh-TDH in complex with 6-nt dsDNA.\u003cstrong\u003e c,\u003c/strong\u003e Superposition of dsDNA in complex I (white cartoon) and complex III (blue cartoon). \u003cstrong\u003ed,\u003c/strong\u003e Superposition of dsDNA in complex I (10 base pairs, white cartoon) and complex IV (6 base pairs, green cartoon), showing the 45° rotation of the helix due to variations in DNA length. \u003cstrong\u003ee. \u003c/strong\u003eComplex V: crystal structure of Gh-TDH in complex with 11-nt dsDNA with 1-nt 3'-overhang. \u003cstrong\u003ef,\u003c/strong\u003e Complex VI: crystal structure of Gh-TDH K88A variant in complex with 11-nt dsDNA with 1-nt 3'-overhang. \u003cstrong\u003eg, \u003c/strong\u003eThe major structural difference between complex III (blue) and complex V (cyan). The movement is marked by three arrows, showing that the C\u003csub\u003e1\u003c/sub\u003e base in complex V moves toward the hydroxyl group of Y87, accompanied by conformational changes in K88 and D89. The sense and antisense strands of the dsDNA are colored green and white, respectively. Hydrogen bonds are depicted as dashed lines, while key binding residues are shown as sticks.\u003cdel\u003e\u003cbr\u003e\n\u003cbr\u003e\n\u003c/del\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/dacf593a6764497e790ebcd5.png"},{"id":77398212,"identity":"ec5c4c5a-28d9-4b64-9222-922a5065db02","added_by":"auto","created_at":"2025-02-28 08:03:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1830344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNuclease activity of wild-type Gh-TDH and its variants. a,\u003c/strong\u003eRelative activities of Gh-TDH variants (W65A, Y87A, K88A, D89A, Y107A, Y87A/Y107A, K88R, K88H, K88E, K88D, K88Q, and K88N) were compared with wild-type (WT) Gh-TDH using a 5'-FAM-labeled stem-loop dsDNA with a 4-nt 3'-overhang.\u003cstrong\u003e \u003c/strong\u003eActivity was measured at 5 mM protein concentration and 0.5 mM substrate concentration. Data are shown as mean ± s.d. (n = 3). \u003cstrong\u003eb,c,\u003c/strong\u003e 20% TBE urea polyacrylamide gel electrophoresis of DNA products after incubation with Gh-TDH WT or its variants.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/68123934b41e9649da4b1553.png"},{"id":77399161,"identity":"8527214b-f8f4-43dc-9819-24f1609c80c7","added_by":"auto","created_at":"2025-02-28 08:11:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6144048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/e155dead-e716-4c78-89cc-c3105485064b.pdf"},{"id":77397772,"identity":"2e02dda4-cd66-416f-99ba-d7612f2cc89c","added_by":"auto","created_at":"2025-02-28 07:55:03","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8133439,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/ba767207bba55cfa70b4bbfd.docx"},{"id":77397763,"identity":"60916468-8dc6-4b31-89a5-1264e0f0ce7a","added_by":"auto","created_at":"2025-02-28 07:55:00","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14838666,"visible":true,"origin":"","legend":"\u003cp\u003e1 (crystal structure data)\u003c/p\u003e","description":"","filename":"structures.zip","url":"https://assets-eu.researchsquare.com/files/rs-5915599/v1/5e8450b7cd0ae35c44cb9882.zip"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Structural and Biochemical Characterization of Grimontia hollisae Thermostable Direct Hemolysin with DNA Reveals First Vibrio Hemolysin with Nuclease Activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePathogenic bacteria produce a range of toxins, including pore-forming toxins (PFTs), cytotoxins, neurotoxins, superantigens, and nucleases, to damage host cells and evade immune responses. For example, \u003cem\u003eVibrio cholerae\u003c/em\u003e cytolysin disrupts cell membranes, causing severe gastrointestinal symptoms, while \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e DNase degrades neutrophil extracellular traps (NETs), aiding immune evasion\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. Group A \u003cem\u003eStreptococcus\u003c/em\u003e (GAS) DNase exacerbates disease by degrading host DNA, reducing pus viscosity, and facilitating bacterial spread\u003csup\u003e4\u003c/sup\u003e. Similarly, \u003cem\u003eMycoplasma bovis\u003c/em\u003e MbovNase degrades NETs and induces apoptosis\u003csup\u003e5\u003c/sup\u003e. Notably, colicin E9 from \u003cem\u003eEscherichia coli\u003c/em\u003e is the first identified bacterial PFT with nuclease activity, enabling DNA damage with the host cytoplasm\u003csup\u003e6,7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThermostable direct hemolysin (TDH) is a PFT and a major virulence factor of pathogenic \u003cem\u003eVibrio\u003c/em\u003e species, including \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (Vp-TDH), \u003cem\u003eV. cholera non-O1, V. mimicus, V. alginolyticus, and Grimontia hollisae\u003c/em\u003e (formally described as \u003cem\u003eV. hollisae\u003c/em\u003e) etc\u003csup\u003e8\u0026ndash;13\u003c/sup\u003e. Ingesting raw seafood or exposure to wounds contaminated with \u003cem\u003eVibrio\u003c/em\u003e TDH can lead to hypovolemic shock, bacteremia, and sepsis\u003csup\u003e14\u0026ndash;22\u003c/sup\u003e. Studies have shown that TDH from \u003cem\u003eG. hollisae\u003c/em\u003e (Gh-TDH) induces osmotic lysis of red blood cells\u003csup\u003e23,24\u003c/sup\u003e, and exhibits cyto-, cardio-, entero-, and hepato-toxicity in cultured cells, as well as lethality in mice\u003csup\u003e16,24\u0026ndash;30\u003c/sup\u003e. Interestingly, while Gh-TDH binds erythrocyte membranes across species with similar affinity, its hemolytic activity varies, suggesting additional activities or factors contribute to its pathogenicity\u003csup\u003e31\u003c/sup\u003e. Additionally, an \"Arrhenius effect\" has been observed, where Gh-TDH is detoxified at 60\u0026ndash;70 \u003csup\u003eo\u003c/sup\u003eC but reactivates above 80 \u003csup\u003eo\u003c/sup\u003eC\u003csup\u003e24,32\u003c/sup\u003e, implicating multiple promoter interfaces in its oligomerization. Crystal structure analysis reveals three distinct tetrameric oligomeric conformations of Gh-TDH: oligomer I, similar to Vp-TDH, and oligomers II and III, which share a dimer motif. This dimer likely represents the minimal structural unit required for membrane binding and hemolytic activity, highlighting the structural complexity of Gh-TDH's role in pathogenesis\u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIncubation of fluorescent recombinant Gh-TDH-FITC with liver cells demonstrated that Gh-TDH binds to the cell membrane, induces extensive membrane blebbing, translocates to the nucleus, and triggers cell lysis and death\u003csup\u003e16\u003c/sup\u003e. This cytotoxic mechanism shares similarities with glucocorticoids and toxic compounds, which induce apoptosis through activation of intracellular receptors that translocate to the nucleus, regulate gene expression, and activate endonucleases\u003csup\u003e33\u003c/sup\u003e. These findings suggest that Gh-TDH may enhance its virulence by either activating endogenous nuclease-dependent apoptosis pathways or directly cleaving DNA through intrinsic nuclease activity, thereby amplifying its cytotoxic potential\u003csup\u003e34\u0026ndash;36\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis study investigated whether Gh-TDH directly degrades cellular DNA or activates endogenous nucleases. \u003cem\u003eIn vitro\u003c/em\u003e nuclease activity assays and X-ray crystallography revealed the structural and mechanistic basis of Gh-TDH's nuclease activity. The assays demonstrated that Gh-TDH binds DNA and catalyzes 3'\u0026ndash;5' exonucleolysis, producing a 3-mer product. Structure analysis of the Gh-TDH-DNA complexs revealed a unique DNA-binding and cleavage mechanism, as Gh-TDH lacks the canonical catalytic motifs typical of 3'\u0026ndash;5' exonucleases, underscoring its distinct nuclease properties\u003csup\u003e37\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of 3'\u0026ndash;5' exonuclease activity of Gh-TDH\u003c/h2\u003e \u003cp\u003eRecombinant wild-type and mutant Gh-TDH proteins were expressed and produced in \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)pLysS and purified for biochemical and structural studies. To evaluate nuclease activity and substrate specificity, various DNA and RNA substrates, including 5'- or 3'-FAM or biotin-labeled single- and double-stranded DNAs, stem-loop dsDNA or dsRNA with varying 5'- or 3'-overhang lengths, and poly(dA\u003csub\u003e12\u003c/sub\u003e), poly(dT\u003csub\u003e12\u003c/sub\u003e), and poly(dC\u003csub\u003e12\u003c/sub\u003e), were synthesized (Supplementary Table\u0026nbsp;1). Gh-TDH efficiently digested 5'-FAM-labeled stem-loop DNA in a concentration-dependent manner, producing a 3-nt 5'-FAM-labeled fragment through 3'\u0026ndash;5' exonucleolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, lanes 1\u0026ndash;5 and 11\u0026ndash;15). However, activity on 3'-FAM-labeled stem-loop substrates was significantly reduced, with cleavage of the FAM tag observed only at high enzyme concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, lanes 6\u0026ndash;10 and 16\u0026ndash;20). When incubated with 5'-FAM-labeled stem-loop dsDNA containing varying 3'-overhang lengths, Gh-TDH digested the overhangs to form blunt-ended dsDNA intermediates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These results indicate that Gh-TDH preferentially trims single-stranded 3'-overhangs in a 3'\u0026ndash;5' exonucleolytic manner before processing dsDNA stem-loops. The 3'-FAM tag inhibits nuclease activity, reducing cleavage efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Regardless of overhang length, Gh-TDH processes DNA until a blunt-ended dsDNA structure is formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), highlighting its substrate preference for 3'-overhangs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGh-TDH's ribonuclease activity was evaluated using 5'-FAM-labeled stem-loop dsRNA with 4-nt 3'-overhang. The enzyme cleaved RNA but exhibited\u0026thinsp;~\u0026thinsp;70% lower efficiency compared to its deoxyribonuclease activity (Supplementary Fig.\u0026nbsp;1). Substrate specificity studies using 5'-FAM-labeled poly(dA\u003csub\u003e12\u003c/sub\u003e), poly(dT\u003csub\u003e12\u003c/sub\u003e), and poly(dC\u003csub\u003e12\u003c/sub\u003e) showed that Gh-TDH efficiently degraded poly(dA\u003csub\u003e12\u003c/sub\u003e) and poly(dT\u003csub\u003e12\u003c/sub\u003e) in a time-dependent manner, with significantly reduced activity on poly(dC\u003csub\u003e12\u003c/sub\u003e). The cleavage produced final 3-nt fragments, resulting in poly(dA\u003csub\u003e3\u003c/sub\u003e), poly(dT\u003csub\u003e3\u003c/sub\u003e), and poly(dC\u003csub\u003e3\u003c/sub\u003e) products (Supplementary Fig.\u0026nbsp;2). The influence of divalent metal ions on Gh-TDH's 3'\u0026ndash;5' exonuclease activity was also investigated. Mg\u003csup\u003e2+\u003c/sup\u003e and Co\u003csup\u003e2+\u003c/sup\u003e significantly enhanced activity, Mn\u003csup\u003e2+\u003c/sup\u003e exerted a marginal effect, while Ca\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, and Zn\u003csup\u003e2+\u003c/sup\u003e showed no enhancement (Supplementary Fig.\u0026nbsp;3a). Analysis of Mg\u003csup\u003e2+\u003c/sup\u003e concentrations showed that Gh-TDH exhibited optimal activity at 5\u0026ndash;10 mM, while higher concentrations leading to a dose-dependent decline (Supplementary Fig.\u0026nbsp;3b). In addition, ethylenediaminetetraacetic acid (EDTA) inhibited nuclease activity, confirming the requirement of divalent metal ions for Gh-TDH function (Supplementary Fig.\u0026nbsp;3b). Additionally, the final DNA cleavage product matched the size of the synthetic 3-mer sequence CTA (Supplementary Fig.\u0026nbsp;3c).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOverall structure of Gh-TDH-DNA complexes\u003c/h3\u003e\n\u003cp\u003eTo investigate how Gh-TDH processes DNA, we determined its crystal structure bound to a 12-nt ssDNA using molecular replacement with the Gh-TDH structure (PDB entry: 4WX3)\u003csup\u003e30\u003c/sup\u003e. Crystallization conditions, data collection, and refinement statistics are detailed in Supplementary Tables\u0026nbsp;2 and 3. Two Gh-TDH-DNA complexes (complexes I and II), differing in \u003cem\u003eN\u003c/em\u003e-terminal conformation, were obtained under distinct crystallization conditions, both with identical lattice packing and space group (P42\u003csub\u003e1\u003c/sub\u003e2). In both complexes, eight ssDNA strands form four duplexes, each comprising 10 base-paired (C\u003csub\u003e3\u003c/sub\u003e\u0026ndash;G\u003csub\u003e12\u003c/sub\u003e) nucleotides and a 2-nt unpaired 5'-overhang. One end of the duplexes binds the same Gh-TDH tetramer, while the other end associates with protomers from four different tetramers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). Complex I showed disordered \u003cem\u003eN\u003c/em\u003e-terminal residues 1\u0026ndash;11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,c), while in complex II, residues 7\u0026ndash;11 are ordered and inserted into the tetramer's central pore (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,c). Structure superposition reveals a conformational shift starting at Asp14 (D14), with a β-sheet stabilized by Gly62 (G62) and D14 in complex I replaced by a loop in complex II (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Despite \u003cem\u003eN\u003c/em\u003e-terminal differences, the DNA conformation remains unchanged, suggesting \u003cem\u003eN\u003c/em\u003e-terminal region variability do not affect DNA binding. Both complexes exhibited a narrowed central pore (19 \u0026Aring; vs. 20 \u0026Aring; in Gh-TDH) and a slight structural adjustment, with complex I showing a 5\u003csup\u003eo\u003c/sup\u003e rotation along the x-axis, resulting in a root mean square deviation (r.m.s.d.) of 1.54 \u0026Aring; relative to the Gh-TDH structure (Supplementary Fig.\u0026nbsp;4). These findings indicate minor conformational changes in the Gh-TDH tetramer accommodate binding of four duplexes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe crystal structures of complexes I and II revealed that Tyr87 (Y87) of the Gh-TDH protomer caps the 5'-end of each DNA duplex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In Gh-TDH, residues Y87\u0026ndash;G90 form a β-hairpin structure between the sixth and seventh β-strands (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Upon DNA binding, these residues undergo conformational changes to interact with the C\u003csub\u003e3\u003c/sub\u003e-G\u003csub\u003e12\u003c/sub\u003e base pair (C\u003csub\u003e3\u003c/sub\u003e on the sense strand and G\u003csub\u003e12\u003c/sub\u003e on the anti-sense strand) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and Supplementary Fig.\u0026nbsp;4b). Key interactions include the indole side chain of Trp65 (W65), which forms a 2.8 \u0026Aring; hydrogen bond with the carbonyl group of C\u003csub\u003e3\u003c/sub\u003e, and the phenolic ring of Y87, which engages in π-π stacking (3.9 \u0026Aring;) with C\u003csub\u003e3\u003c/sub\u003e's aromatic pyrimidine ring. In the β-turn, the main chain amino group of Asp89 (D89) hydrogen bonds with the N7 amine (2.8 \u0026Aring;) and carbonyl group (3.4 \u0026Aring;) of G\u003csub\u003e12\u003c/sub\u003e, while the amino group of Gly90 (G90) forms a 3.0 \u0026Aring; hydrogen bond with G\u003csub\u003e12\u003c/sub\u003e's carbonyl group. Additionally, the side chains of Lys88 (K88) and D89 create water-mediated hydrogen bonds with the phosphate backbone between G\u003csub\u003e11\u003c/sub\u003e and G\u003csub\u003e12\u003c/sub\u003e. In complex I, interactions between K88, D89, and the 3'-end of the dsDNA stabilize their structural flexibility. This stabilization is reflected in the reduced B-factors for K88's amino group in Gh-TDH compared to that in complex I (52.9 to 21.0). The elasticity of residues Y87\u0026ndash;G90's β-hairpin structure facilitates Gh-TDH's capacity to adopt and bind diverse DNA structures effectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eGh-TDH recognizes different dsDNA substrates in a similar mode\u003c/h3\u003e\n\u003cp\u003eTo evaluate the impact of DNA sequence and length on Gh-TDH's binding and cleavage activity, 10-nt and 6-nt DNA substrates (with or without 5'-FAM-labels) were used in nuclease assays and structural analysis (complexes III and IV). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, Gh-TDH cleaved both 10-nt and 6-nt DNAs in a concentration-dependent manner. Cleavage sites were identified through high-resolution mass analysis (HD Q-TOF) of a 6-nt DNA substrate with hydroxyl groups at both ends (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The analysis revealed a molecular mass of 845.2002, matching the calculated mass of a CTA 3-mer with a 3'-hydroxyl group (Mw\u003csub\u003ecalc\u003c/sub\u003e.: 845.2015) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), confirming that Gh-TDH cleaves 3' O-P bond to produce a 3-mer DNA with 3'-hydroxyl group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eStructural analysis of complexes III and IV revealed that the 10-nt and 6-nt ssDNA substrates used for co-crystallization are self-complemented, forming blunt-ended dsDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). The \u003cem\u003eN\u003c/em\u003e-terminal residues Pro7\u0026ndash;Pro11, visible in complexes II and III, were absent in complex IV, where the \u003cem\u003eN\u003c/em\u003e-terminus remained disordered, resembling complex I and the Gh-TDH structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). Despite differences in DNA length and sequence (A\u003csub\u003e9\u003c/sub\u003e and A\u003csub\u003e5\u003c/sub\u003e in complexes III and IV, vs. G\u003csub\u003e11\u003c/sub\u003e in complex I) (Supplementary Table\u0026nbsp;2), the binding modes in complexes III and IV closely resemble that of complex I (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b). In complex III, the indole side chain of W65 forms a hydrogen bond with C\u003csub\u003e1\u003c/sub\u003e's carbonyl group (2.9 \u0026Aring;, sense strand), Y87's phenolic side chain stacks with C\u003csub\u003e1\u003c/sub\u003e (3.7 \u0026Aring;), and D89's main chain amino group forms hydrogen bonds with G\u003csub\u003e10\u003c/sub\u003e's (antisense strand) N7 amine (2.8 \u0026Aring;) and carbonyl group (3.5 \u0026Aring;). G90's main chain amino group forms hydrogen bonds with A\u003csub\u003e9\u003c/sub\u003e's N7 amine (3.5 \u0026Aring;) and G\u003csub\u003e10\u003c/sub\u003e's carbonyl group (2.9 \u0026Aring;) in complex III (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), compared to a hydrogen bond with only the G\u003csub\u003e12\u003c/sub\u003e's carbonyl group (2.9 \u0026Aring;) in complex I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Complex IV shows a nearly identical DNA-binding environment to complex III (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), with consistent interaction regions across the Gh-TDH-DNA complexes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). In addition, variations in length result only in a shift of the DNA helix, as evidenced by the 45\u0026deg; rotation of the helix between complex I and complex IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). This result suggests that Gh-TDH binds to dsDNA similarly, regardless of DNA length and sequence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eLys88 is an important residue for DNA cleavage\u003c/h3\u003e\n\u003cp\u003eAlanine scanning mutagenesis of Gh-TDH residues near the DNA-binding site (W65, Y87, K88, D89, Y107A, and Y87/Y107) was performed to assess their role in nuclease activity using a 5'-FAM-labeled stem-loop dsDNA with a 4-nt 3'-overhang. The K88A variant showed the most significant reduction, completely losing exonuclease activity at 5 \u0026micro;M protein concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,b). Mutations of Y87A, D89A, and Y107A had minimal impact, while W65A and Y87A/Y107A caused partial reductions. Further testing of K88 mutants (K88E, K88D, K88Q, K88N, K88R, and K88H) showed that K88E, K88D, K88Q, and K88N reduced activity to 66%, 49%, 49%, and 54%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea,c), while K88R retained wild-type activity and K88H showed a slight increase. These results suggest that a positively charged residue at position 88 is important for DNA cleavage. Dissociation constants (\u003cem\u003eK\u003c/em\u003e\u003csub\u003eD\u003c/sub\u003e) for wild-type Gh-TDH and the K88A variant, measured by bio-layer interferometry (BLI), were 14.8 and 22.4 \u0026micro;M, respectively, indicating that the loss of exonuclease activity in the K88A variant is not attributable to changes in substrate binding affinity (Supplementary Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effect of K88 mutation on DNA binding and trimming, wild-type Gh-TDH and the K88A variant were co-crystallized with an 11-nt ssDNA, forming complex V and complex VI, respectively (Supplementary Tables\u0026nbsp;2 and 3). In solution, the two ssDNA strands form a dsDNA with a 1-nt 3'-overhang and a 10-nt duplex. In both complexes, the first 11 \u003cem\u003eN\u003c/em\u003e-terminal residues are disordered, as seen in Gh-TDH and complex I (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee,f). Crystal structures revealed that A\u003csub\u003e11\u003c/sub\u003e of the antisense strand forms sugar-π and π-π interactions with W65 and Y107, respectively, differing from the C\u003csub\u003e3\u003c/sub\u003e/G\u003csub\u003e12\u003c/sub\u003e, C\u003csub\u003e1\u003c/sub\u003e/G\u003csub\u003e10\u003c/sub\u003e, and C\u003csub\u003e1\u003c/sub\u003e/G\u003csub\u003e6\u003c/sub\u003e base pairs observed in complexes I-IV (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In addition, Y87 still forms π-π stacking with C\u003csub\u003e1\u003c/sub\u003e, but C\u003csub\u003e1\u003c/sub\u003e shifts closer to the hydroxyl group of Y87 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eIn complexes V and VI, D89 forms a hydrogen bond with the N7 amine of G\u003csub\u003e10\u003c/sub\u003e (the penultimate base), similar to its interaction with the final G base in complexes I-IV. However, in complexes V and VI, G90 forms a hydrogen bond with the N7 amine of G\u003csub\u003e10\u003c/sub\u003e (the penultimate G), unlike in complexes I-IV, where G90 forms a hydrogen bond with the carbonyl group of the final G (G\u003csub\u003e12\u003c/sub\u003e in complexes I and II, G\u003csub\u003e10\u003c/sub\u003e in complex III, G\u003csub\u003e6\u003c/sub\u003e in complex IV) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Additionally, G90's carbonyl group forms a hydrogen bond with the C-4 amino group of C\u003csub\u003e1\u003c/sub\u003e (sense strand) in complexes V and VI, a bond absent in complexes I\u0026ndash;IV. Water molecules bound to K88 and D89 in complexes I-III are not observed in complexes IV-VI (Supplementary Fig.\u0026nbsp;6a\u0026ndash;f). Structural analysis of complexes V and VI, along with BLI results, supports that the K88A variant does not affect Gh-TDH's binding affinity or mode, reinforcing K88's role as a catalytic residue. The differences in binding patterns between complexes III and V suggest that Gh-TDH adopts to various DNA structures to facilitate cleavage. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to identify Gh-TDH, a hemolysin in \u003cem\u003eVibrio\u003c/em\u003e species, with nuclease activity, revealing its distinct structural basis compared to other known exonucleases. Gh-TDH shows no significant sequence or structural similarity to known nucleases. Unlike well-characterized 3'\u0026ndash;5' exonucleases or other nucleases (including the DEDD and DEK superfamilies), which typically use aspartate (Asp) or glutamate (Glu) residues to coordinate divalent metal ions and stabilize the transition state during phosphodiester bond cleavage\u003csup\u003e37,38\u003c/sup\u003e, Gh-TDH primarily relies on aromatic residues Trp65 and Tyr87 to interact with the non-scissile strand. Structural analysis shows that members of the DEDD superfamily, such as ExoX and TREX2, utilize a loop between β1 and β2 strands or α-helical loop structure to specifically bind to the non-scissile strand of dsDNA\u003csup\u003e39,40\u003c/sup\u003e. Additionally, while the general base in DEDD family enzymes is typically located within a loop (e.g., TREX2's α6\u0026ndash;α7 loop)\u003csup\u003e37\u003c/sup\u003e, in Gh-TDH, the general base Lys88 is located within a unique β-turn (K88 to G90) which plays a critical role in DNA binding and cleavage.\u003c/p\u003e \u003cp\u003eGh-TDH is a metal ion-dependent nuclease that lacks the typical conserved motif of 3'\u0026ndash;5' exonucleases. Mutational analysis of acidic residues near the DNA-binding site (D89, D117, E118) showed no significant effect on nuclease activity (Supplementary Fig.\u0026nbsp;6g), indicating these residues may play supporting roles in coordinating the divalent metal ion. While most metal-dependent nucleases use a bimetallic ion mechanism, monometallic ion mechanisms are more common in endonucleases\u003csup\u003e37\u003c/sup\u003e. Notably, in the bimetallic ion nuclease Ape1 (PDB entry: 7CD5), the divalent metal ions were not determined in the crystal structure and was proposed to be captured in its product-bound form\u003csup\u003e41\u003c/sup\u003e. The metal ion dependency of Gh-TDH, coupled with the absence of detectable divalent ions in the Gh-TDH-DNA complex, suggests the complex may also be in a product-bound state. In most nucleases, histidine or tyrosine residue near the active site usually functions as a general base, activating water molecules to initiate DNA cleavage\u003csup\u003e37\u003c/sup\u003e. However, mutations of Y87 and Y107 in Gh-TDH had little effect on activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, mutations at K88 significantly affected activity. Structural analysis revealed that K88 interacts with the DNA 3'-end through a water-mediated network involving water molecules W\u003csub\u003e2\u003c/sub\u003e, W\u003csub\u003e3\u003c/sub\u003e, and W\u003csub\u003e4\u003c/sub\u003e (Supplementary Fig.\u0026nbsp;6a\u0026ndash;c). This function is analogous to the role of K131 in the bacteriophage lambda exonuclease as a general base\u003csup\u003e42\u003c/sup\u003e. K88 in Gh-TDH likely plays a central role as a general base for activating water molecules during the catalytic process.\u003c/p\u003e \u003cp\u003eStructural comparison of complexes I\u0026ndash;VI revealed only minor conformational changes in the hydrophobic pocket residues Y87, W65, and Y107, despite variations in DNA binding. A consistent π-π stacking interaction between Y87 and the first cytosine (sense strand) was observed across all complexes, with slight conformational shifts in complexes V and VI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). In complexes I\u0026ndash;IV, W65 forms a hydrogen bond with the first cytosine (sense strand), while in complexes V and VI, a sugar-π interaction with A\u003csub\u003e11\u003c/sub\u003e (antisense strand) is observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Y107 interacts minimally with DNA in complexes I\u0026ndash;IV, but forms a π-π interaction with A\u003csub\u003e11\u003c/sub\u003e (antisense strand) in complexes V and VI. These shifts likely enable Gh-TDH to distinguish between scissile and non-scissile strands, facilitating precise cleavage at the 3'-end of the ssDNA substrate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). The reduced exonuclease activity of the W65A and Y87A/Y107A variants further highlights the importance of these residues in efficient DNA cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Similar aromatic residues in RNase T (Phe29) and TREX2 (Tyr129), members of the DEDD 3'\u0026ndash;5' exonuclease superfamily, also play key roles in precise 3'-overhang cleavage\u003csup\u003e39,40,43\u0026ndash;45\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Gh-TDH-DNA complexes revealed the structure of five additional amino acids (\u003csup\u003e7\u003c/sup\u003ePFPAP\u003csup\u003e11\u003c/sup\u003e) at the \u003cem\u003eN\u003c/em\u003e-terminus, a region critical for hemolytic activity and pore formation in Gh-TDH and Vp-TDH\u003csup\u003e30,46,47\u003c/sup\u003e. Despite its importance, the mechanism of pore formation remains unclear, as the \u003cem\u003eN\u003c/em\u003e-terminal region is disordered in both TDH crystal structures. Cryo-EM analysis of Vp-TDH shows NTR density maps associated with pore-forming structures, but its exact role in pore formation is yet to be determined\u003csup\u003e48\u003c/sup\u003e. The complex structures determined in this study provide evidence that the \u003cem\u003eN\u003c/em\u003e-terminal region undergoes two distinct conformational changes during the pore-forming process. Our findings highlight the presence of these additional amino acids (\u003csup\u003e7\u003c/sup\u003ePFPAP\u003csup\u003e11\u003c/sup\u003e) in complexes II and III, which may provide valuable insights into the functional significance of NTR.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eNucleases are vital for DNA repair and genome integrity, with human nucleases like TREX1 and TREX2 maintaining stability\u003csup\u003e40,45,49\u003c/sup\u003e. In contrast, pathogenic bacterial nucleases, such as Cdt, can enter the host nucleus, targeting host DNA and disrupting host nucleases, leading to DNA damage and cell death\u003csup\u003e50\u003c/sup\u003e. This study identifies Gh-TDH as the first \u003cem\u003eVibrio\u003c/em\u003e hemolysin with nuclease activity. Through \u003cem\u003ein vitro\u003c/em\u003e assays, mass spectrometry, structural analysis, and mutagenesis, we confirm Gh-TDH as a novel 3'\u0026ndash;5' exonuclease with both hemolytic and nuclease functions. These findings lay the groundwork for targeted therapeutic strategies against \u003cem\u003eVibrio\u003c/em\u003e infections, with future research focusing on Gh-TDH's membrane interactions and permeabilization.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eMolecular cloning, mutagenesis, gene expression, protein production and purification of the recombinant Gh-TDH wild-type and the mutant variants\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe Gh-TDH gene (GenBank accession ID: WP_040528653.1) was cloned into the pCR2.1-TOPO vector. For the mutant variants, the plasmids were constructed by the QuikChange site-directed mutagenesis method. All plasmids were confirmed via DNA sequencing. Each of the wild-type and the mutant constructs was transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3)pLysS cells for gene expression. Gene expression, protein production, and purification were carried out using previously established methods, yielding highly purified proteins for biochemical and structural analysis\u003csup\u003e24,29\u003c/sup\u003e. The protein purity was assessed by SDS-PAGE. All wild-type and the mutant variants of Gh-TDH proteins were concentrated using Amicon Ultra-15 3,000 NMWL concentrator (Merck) in 20 mM Tris-HCl at pH 7.0 for the following nuclease activity assay, BLI analysis, and protein crystallization.\u003c/p\u003e\n\u003ch3\u003eNuclease activity assay\u003c/h3\u003e\n\u003cp\u003eFluorescein amidite (FAM)-labeled DNA substrates (3'- or 5'-end) were synthesized by Genomics Inc. Taiwan for nuclease activity assays. The sequences of DNA substrates used in nuclease activity are listed in Supplementary Table\u0026nbsp;1. In nuclease activity assays, 0.5 \u0026micro;M DNA substrates were incubated with protein in 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e and 20 mM Tris-HCl (pH 7.0). After incubation at 37 ℃ for 60 min, 2x TBE-urea loading buffer (G-Biosciences) was added to quench the traction at 95 ℃ for 5 min. DNA digestion patterns were analyzed using 20% TBE urea polyacrylamide gels and visualized under blue light. Nuclease-specific activity was measured using 5 \u0026micro;M protein with 0.5 \u0026micro;M substrate, with excision quantified using ImageJ.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCrystallization, data collection, and structure determination\u003c/h2\u003e \u003cp\u003eThe purified Gh-TDH wild-type, and K88A variant were mixed with ssDNA at a 1:1.3 molar ratio, respectively, and incubated on ice for 20 minutes. The mixtures were crystallized using the hanging-drop vapor-diffusion method at 20\u0026deg;C. The crystallization conditions and the sequences of co-crystallized ssDNA for complexes I\u0026ndash;VI were listed in Supplementary Table\u0026nbsp;2. Crystals were cryo-protected with Paraton-N (Hampton Research) for data collection at BL15-A1, TPS-05A, and TPS-07A (NSRRC, Taiwan, R.O.C.). All diffraction data were indexed and scaled with HKL2000\u003csup\u003e51\u003c/sup\u003e. Molecular replacement was performed using Gh-TDH (PDB entry: 4WX3) as the search model\u003csup\u003e30\u003c/sup\u003e in MOLREP of CCP4\u003csup\u003e52\u003c/sup\u003e. Models were built in COOT\u003csup\u003e53\u003c/sup\u003e and refined with REFMAC\u003csup\u003e54\u003c/sup\u003e. The atomic coordinates and structure factors of complexes I\u0026ndash;VI have been deposited in the Protein Data Bank with the accession code 9LCS, 9L8E, 9L9M, 9LCM, 9LCU, and 9LCW, respectively. Data processing and refinement statistics are summarized in Supplementary Table\u0026nbsp;3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometric analysis of Gh-TDH DNA cleavage products\u003c/h2\u003e \u003cp\u003eMass spectrometric analyses were performed by the Center for Advanced Instrumentation and Department of Applied Chemistry at National Yang Ming Chiao Tung University, Hsinchu, Taiwan, R.O.C. To analyze the molecular mass of DNA products cleaved by Gh-TDH, a 6-mer DNA substrate (CTATAG) (50 \u0026micro;M) was incubated with Gh-TDH (50 \u0026micro;M) in 20 mM Tris-HCl (pH 7.0) and 5 mM MgCl\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C for 4 hours. To quench the reaction, the mixture was heated to 60\u0026deg;C for 10 min. The reaction mixture was centrifuged at 13,000 rpm to remove the fibrillar Gh-TDH and the supernatant was subjected to HD Q-TOF with ESI(+)MS analysis (Bruker). The collected data were analyzed using Compass DataAnalysis 4.1 (Bruker).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBio-layer interferometry (BLI) binding kinetics assay\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe binding affinity of 5'-biotinylated stem-loop dsDNA to Gh-TDH wild-type or the K88A variant was measured using the Octet HTX system (FroteBio) at the Center for Emergent Functional Matter Science, National Yang Ming Chiao Tung University, Taiwan, R.O.C. BLI measurements were carried out at a shaking speed of 1,000 rpm. The purified Gh-TDH proteins at various concentrations of 10, 20, 30, 40, and 50 \u0026micro;M were prepared in kinetic buffer (PBS at pH 7.4, 50 mM EDTA, 1 mg/mL BSA) and loaded onto HIS1K (streptavidin) biosensors (Molecular Devices, ForteBio). Five concentrations of Gh-TDH in kinetic buffer were added to a black polypropylene 96-well microplate (Greiner Bio-one), with one row containing kinetic buffer as a reference control. Each protein concentration underwent an assay cycle using streptavidin-5'-biotinylated stem-loop dsDNA probes and blank probes. One assay cycle consists of 60 s of baseline normalization in kinetics buffer, 400 s of association in the protein solution, 200 s of dissociation in kinetics buffer, and 30 s of regeneration in 0.5 M NaOH. BLI results were analyzed using Fort\u0026eacute;Bio Data Analysis High Throughput 12.0. The sequences of ssDNA substrates used in BLI assays are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe data associated with this study are available within the article and Supplementary Information. All the coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 9LCS, 9L8E, 9L9M, 9LCM, 9LCU, and 9LCW. Source data are provided with this paper. All other data are available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science and Technology Council (NSTC), Taiwan (grants 110-2113-M-A49-026-MY3, 113-2113-M-A49-025-, and 113-2113-M-A49-018-). This work is also supported in part by the Kaohsiung Medical University Research Center Grant (NYCUKMU-114-I005) and the Center for Emergent Functional Matter Science and the Center for Intelligent Drug Systems and Smart Biodevices (IDS\u003csup\u003e2\u003c/sup\u003eB) of National Yang Ming Chiao Tung University from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan. The authors thank the experimental facility and the technical service provided by the Synchrotron Radiation Protein Crystallography Facility of the National Core Facility Program for Biotechnology and the National Synchrotron Radiation Research Center (NSRRC), a national user facility supported by NSTC, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.Y.H., T.K.W., and C.Y.C. conceived the project, designed the experiments, and wrote the manuscript; P.Y.H., Y.K.W., and S.C.H. performed molecular cloning, protein production, and protein purification; P.Y.H., F.P.C., and T.Y.H. performed binding kinetics and BLI analysis; P.Y.H., Y.C.L., and Y.M.K. performed protein crystallization, diffraction data collection, and structural determination; P.Y.H. performed nuclease activity assay and mass spectrometric analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMondal, A. 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Refinement of macromolecular structures by the maximum-likelihood method. \u003cem\u003eActa Crystallogr D Biol Crystallogr\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 240-255 (1997).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Grimontia hollisae, thermostable direct hemolysin, pore-forming toxin, exonuclease activity, binding and internalization","lastPublishedDoi":"10.21203/rs.3.rs-5915599/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5915599/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eGrimontia hollisae\u003c/em\u003e thermostable direct hemolysin (Gh-TDH) is a pore-forming toxin that disrupts cell membrane, causing erythrocyte lysis and exhibiting cytotoxicity, cardiotoxicity, enterotoxicity, hepatotoxicity, and lethality in mice. Recombinant Gh-TDH-FITC binds to hepatocyte membranes and translocates to the nucleus. This study reveals that Gh-TDH cleaves DNA with 3'\u0026ndash;5' nuclease activity to generate a 3-mer fragment, while cleaving RNA with ~\u0026thinsp;70% lower efficiency. The co-crystal structure of Gh-TDH bound to ssDNA unveils a unique DNA-binding configuration, where eight ssDNA strands form four duplexes, each connected to a Gh-TDH tetramer at one end and to protomers from four different tetramers at the other. Notably, the cleavage site (Tyr87-Lys88-Asp89) deviates from the canonical 3'\u0026ndash;5' exonuclease motif (Asp-Glu-Asp-Asp). Mutagenesis identified Lys88 as a general base essential for nuclease activity. This study provides structural and functional insights of Gh-TDH on DNA-binding and cleavage mechanism. In summary, Gh-TDH is the first pore-forming toxin identified in the \u003cem\u003eVibrio\u003c/em\u003e genus with dual functions of hemolytic and nuclease activities.\u003c/p\u003e","manuscriptTitle":"Structural and Biochemical Characterization of Grimontia hollisae Thermostable Direct Hemolysin with DNA Reveals First Vibrio Hemolysin with Nuclease Activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-28 07:54:55","doi":"10.21203/rs.3.rs-5915599/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1137b8c1-87e5-432b-9519-c884e05f2625","owner":[],"postedDate":"February 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44299165,"name":"Biological sciences/Biochemistry/DNA"},{"id":44299166,"name":"Biological sciences/Biochemistry/Structural biology/X-ray crystallography"},{"id":44299167,"name":"Biological sciences/Biochemistry/Enzymes"}],"tags":[],"updatedAt":"2025-02-28T07:54:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-28 07:54:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5915599","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5915599","identity":"rs-5915599","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}