Elucidating the Molecular Mechanism of 3D1 Antibody Binding to a Swine Enteric Coronavirus Antigen

TL;DR

This study reveals how the 3D1 antibody neutralizes a swine coronavirus by binding to a specific viral protein structure, preventing the virus from fusing with host cells.

Contribution

The study identifies a conserved epitope and mechanism by which 3D1 traps a pre-hairpin intermediate state of the viral spike protein.

Findings

3D1 binds to a linear β-turn motif in the HR1 domain of the viral spike protein with high affinity.

3D1 competitively inhibits the interaction between HR1 and HR2, blocking the formation of the postfusion six-helix bundle.

3D1 maintains strong binding to HR1 even when it is in a conformationally constrained, helical state.

Abstract

The broadly neutralizing monoclonal antibody 3D1 potently neutralizes SADS-CoV by targeting a conserved epitope within the heptad repeat 1 (HR1) domain of the viral spike protein. Structural and biophysical analyses demonstrate that 3D1 binds with high affinity to a specific linear β-turn motif (residues A804–N809) in HR1. High-resolution crystallography reveals that this motif sits within a deep, electrostatically complementary paratope groove. Critically, 3D1 binding competitively inhibits the essential interaction between HR1 and HR2. Notably, its recognition is not dependent on HR1’s native helical conformation, as it maintains strong binding to conformationally constrained, stapled helical peptides. Collectively, the data indicate that 3D1 neutralizes by capturing a pre-hairpin intermediate state of HR1—a transition state between prefusion and postfusion forms—thereby sterically blocking the formation of the stable postfusion six-helix bundle that is essential for membrane fusion. This work defines a precise, structure-dependent neutralizing epitope and elucidates a mechanism of action that involves trapping a key fusion intermediate, offering a valuable template for the design of broad-spectrum coronavirus therapeutics.

Figures

Binding modes and intermolecular interactions of the protein–peptide complex. (a) The refined conformation of the pepAVVNQN motif from the crystal structure. The conformation enclosed by the dashed circle is atypical and differs from the canonical type I β-turn, as compared to the previously resolved 3D1-pepDVVNQN structure. (b) Conformational alignment of the peptide from the molecular dynamics trajectory, demonstrating high structural overlap. (c) Principal component analysis (PCA) of the peptide trajectory, highlighting the three dominant conformational states sampled. Spot A represents the most populated cluster. Spot B and Spot C correspond to distinct, metastable conformational states, illustrating the key dynamic transitions within the trajectory. (d) Network of key intermolecular interactions stabilizing the complex. Hydrogen bonds are shown as green dashed lines, and hydrophobic contacts are indicated by spoked arcs. The figure was generated using the LigPlot+ tool [30]. Atom colors are as follows: carbon (black), oxygen (red), and nitrogen (blue). Residues in green mediate interactions with pepAVVNQN, whereas hydrophobic interactions are represented by the spoked arcs. H and L denote the heavy and light chains of antibody 3D1, respectively. The pepAVVNQN is shown in purple. Notably, residue N35 forms a hydrogen bond with an occupancy of 97.0%. (e) Time evolution of the van der Waals (VDW) and electrostatic (ELE) interaction energies between the peptide and protein. (f) B-factor putty representation of the protein derived from RMSF analysis, indicating low flexibility (thin tubes) for most residues surrounding the bound peptide (magenta sticks).

Putative mode of action of 3D1 and binding to constrained peptides. (a,b) Design and binding analysis of stapled peptides. Left panels: Schematics of the two stapling strategies: (a) all-hydrocarbon staple via ring-closing metathesis; (b) azo-bridge crosslink. Right panels: Binding curves from competition assays showing that 3D1 retains strong binding to both stapled peptides (α-helical content > 80%). RCM, ring-closing metathesis; azo, azobenzene. The light-blue text and stick models designate residues that are either non-natural or carry an AZO modification on their side chains. (c) Isothermal titration calorimetry (ITC) data confirming a 1:1 binding stoichiometry between 3D1 and the HR1-SADS antigen. The upper panel shows raw heat pulses, and the lower panel shows the integrated binding isotherm fitted to a single-site model. (d) Proposed model for 3D1-mediated neutralization. 3D1 binds to a putative pre-hairpin intermediate conformation of HR1 adopting a type I β-turn, thereby blocking its structural transition and subsequent association with HR2 to form the post-fusion six-helix bundle (PDB: 8X7X) [41]. The β-turn conformation shown here was experimentally observed in antibody-bound HR1-derived peptides and is not directly visualized during the viral fusion process; its assignment as a fusion intermediate is therefore inferred from structural and biochemical data. Similar transient non-helical or β-turn–like intermediates have been reported for other class I viral fusion proteins, supporting the plausibility of this model. The transition from prefusion to post-fusion occurs within minutes of host cell attachment.

Full text

1. Introduction

Coronaviruses pose a persistent threat to global health, underscored by the emergence of SARS-CoV-2 and the continued circulation of animal viruses with zoonotic potential [1]. The spike protein, a class I viral fusion protein, mediates host cell entry and is a primary target for neutralizing antibodies. Its fusion mechanism involves a conformational rearrangement where heptad repeat regions HR1 and HR2 assemble into a stable six-helix bundle (6HB), driving the merger of viral and host membranes. Similar to SADS-CoV, the spike proteins of other coronaviruses, including SARS-CoV-2, contain conserved heptad repeat 1 (HR1) and heptad repeat 2 (HR2) regions within the S2 subunit, which associate to form a six-helix bundle (6-HB) that drives viral and host membrane fusion [2]. The HR1 domain, which forms a central trimeric coiled-coil in the post-fusion state, presents a conserved and vulnerable site for therapeutic intervention.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an alphacoronavirus identified in 2017, causes severe enteric disease in piglets with significant economic impact [3,4]. Its spike protein shares a modular architecture with other coronaviruses, yet the structural details of its fusion machinery and its vulnerability to antibody neutralization [5,6] remain poorly defined. Monoclonal antibody 3D1, previously identified, exhibits neutralizing activity against SADS-CoV [5]. Preliminary evidence suggests it targets the HR1 domain and disrupts its trimeric structure, but the precise molecular basis for this activity is unknown.

Understanding how 3D1 recognizes and inactivates HR1 is critical for defining a key vulnerability in SADS-CoV and potentially related coronaviruses. Here, we combine biochemical assays, X-ray crystallography [7], molecular dynamics simulations, and protein engineering to dissect this interaction [8]. We demonstrate that 3D1 recognizes a specific β-turn epitope within HR1 with high affinity and conserved architecture. Surprisingly, this recognition is maintained even when the native HR1 helical structure is chemically stabilized. Our findings delineate a detailed mechanism of neutralization wherein 3D1 traps a fusion intermediate, providing a structural blueprint for the rational design of cross-reactive antibodies and fusion inhibitors.

2. Materials and Methods

2.1. Peptide Synthesis

A series of biotinylated and non-biotinylated peptides spanning the predicted 3D1 epitope region (residues 748–836 of the SADS-CoV S protein, including the pep^AVVNQN^ peptide with the full sequence A804-V805-V806-N807-Q808-N809) and FITC-HR2 peptide were synthesized commercially by GenScript Biotech (Nanjing, Jiangsu, China). The peptides were provided with a purity of >95%, as verified by high-performance liquid chromatography (HPLC) and mass spectrometry. Upon receipt, lyophilized peptides were reconstituted in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA, #D8418) or PBS to prepare stock solutions, aliquoted, and stored at −80 °C.

2.2. Design and Synthesis of Stapled Peptides

Two stapled peptide variants of the HR1C sequence containing the pep^AVVNQN^ (A804-V805-V806-N807-Q808-N809) motif were designed. The first all-hydrocarbon staple was accomplished by ring-closing metathesis between the olefin-bearing non-natural amino acids R8 (octenylalanine) and S5 (pentenylalanine), incorporated at specific i and i + 7 positions. The second, an azobenzene-crosslinked staple, was formed by coupling cysteine residues flanking the motif with a bifunctional azo-linker. Peptides were synthesized via standard Fmoc solid-phase peptide synthesis, purified by HPLC, and characterized by mass spectrometry. These two peptides were designed and synthesized by GenScript Biotech (Nanjing, Jiangsu, China).

2.3. Cell Culture

Human embryonic kidney (HEK-293F) cells (Thermo Fisher Scientific, Waltham, MA, USA, #R79007) were maintained in FreeStyle 293 Expression Medium (Gibco, Grand Island, NY, USA, #12338018 ) at 37 °C with 8% CO_2_ and 125 rpm agitation for recombinant protein production.

2.4. Plasmid Construct and Protein Expression

The expression plasmids for the 3D1-scFv-His and 3D1-IgG1 proteins were constructed as described before [5]. Briefly, codon-optimized genes for the respective formats were synthesized and cloned into mammalian expression vectors: the scFv construct into a pFUSE-based vector with a C-terminal 6xHis tag, and the Fab-Fc heavy and light chains into the pFUSE-hIgG1-Fc1 (InvivoGen, San Diego, CA, USA, #pfuse-hg1fc1) and pFUSE2-CLIg-hL2 (InvivoGen, San Diego, CA, USA, #pfuse2-hcll2).

For protein expression, plasmids were transfected into HEK-293F cells using polyethylenimine (PEI, linear, MW 40,000, Yeasen, Shanghai, China, #40816ES03). The culture supernatant was harvested 3–4 days post-transfection by centrifugation.

2.5. Protein Purification and Complex Assembly

The 3D1-scFv-His and 3D1-IgG1 were expressed in HEK293F cells and purified by protein A affinity chromatography [9] (Cytiva, Marlborough, MA, USA, #17526801). To generate the 3D1 Fab fragment for complex formation, purified 3D1-IgG1 was digested with papain (Thermo Fisher Scientific, #20341) at a 1:100 (w/w) enzyme-to-substrate ratio in digestion buffer (20 mM sodium phosphate, 10 mM EDTA, 20 mM cysteine-HCl, pH 7.0) at 37 °C for 4 h. The reaction was terminated by adding 30 mM iodoacetamide. The Fab fragment was then separated from the Fc fragment and undigested IgG by reapplying the mixture to the MabSelect SuRe protein A column, which captured the Fc portion and full-length IgG, allowing the Fab fragment to flow through. The flow-through fraction containing the Fab was collected and further purified by size-exclusion chromatography (Superdex 200 Increase 10/300 GL) in PBS, pH 7.4. The complex was formed by incubating the purified Fab with a 1.2 molar excess of peptide.

2.6. Yeast Surface Display and Binding Assays

The SADS-CoV HR1 domain was cloned into the pYD1 vector (Thermo Fisher Scientific, #V83501) for expression as an Aga2p fusion protein on the surface of Saccharomyces cerevisiae EBY100 cells (Thermo Fisher Scientific, #C3030032). Expression was induced following standard protocols. For binding analysis, induced yeast cells were incubated with FITC-labeled HR2 peptide at varying concentrations in PBSA (PBS + 1 mg/mL BSA, Sigma-Aldrich, #A7906) for 1 h at 4 °C. After washing, binding was quantified by flow cytometry (BD FACSCelesta, BD Biosciences, Franklin Lakes, NJ, USA). For competition assays, yeast cells displaying HR1 were pre-incubated with serial dilutions of antibody 3D1 (or buffer control) for 30 min prior to the addition of FITC-HR2 peptide at a concentration near its EC_50_ (~500 nM). Binding signal was measured and normalized to the no-competitor control. To ensure experimental specificity, yeast cells transformed with the empty pYD1 vector and uninduced cells were used as negative controls. Additionally, an isotype-matched irrelevant antibody was employed to exclude non-specific interactions with the yeast cell surface.

2.7. Crystallization and Data Collection

The purified 3D1 Fab–pep^AVVNQN^ complex was concentrated to 12 mg/mL in 20 mM Tris-HCl (pH 8.0, Sigma-Aldrich, #T3253) and 100 mM NaCl (Sigma-Aldrich, #S9888). Crystals were obtained at 20 °C using the sitting-drop vapor-diffusion method [10] by mixing 0.2 μL of protein complex with 0.2 μL of reservoir solution containing 0.1 M sodium citrate tribasic dihydrate (pH 5.5, Hampton Research, Aliso Viejo, CA, USA**, #HR2-623) and 20% (w/v) PEG 3000 (Hampton Research, #HR2-603). Rod-shaped crystals appeared within 3 days and were cryo-protected by brief immersion in reservoir solution supplemented with 20% (v/v) glycerol (Sigma-Aldrich, #G5516) prior to flash-cooling in liquid nitrogen. X-ray diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL19U1 with an exposure time of 0.1 s per frame and a total rotation angle of 180°. A complete dataset was indexed, integrated, and scaled to 2.5 Å resolution using XDS and AIMLESS (from the CCP4 suite) [11].

2.8. Structure Determination and Refinement

The structure was solved by molecular replacement using Phaser within the Phenix suite, with the 3D1 Fab (PDB: 7YI6) serving as separate search models [12]. The initial model was manually rebuilt in Coot to fit the electron density [13], and the pep^AVVNQN^ peptide was built into clear, continuous density within the binding groove. Iterative cycles of refinement using Phenix.refine and manual model adjustment were performed [14]. The final model (R_work_ = 21.2%, R_free_ = 28.1%) includes four Fab–peptide complexes in the asymmetric unit and exhibits good stereochemistry, with 95.2% of residues in the favored regions of the Ramachandran plot. Structural figures were prepared using PyMOL-3.1.4.1 (Schrödinger, LLC, New York, NY, USA).

2.9. Molecular Dynamics Simulations

Molecular dynamics (MD) simulations were performed using GROMACS 2022.2 [8] with a total duration of 500 ns. The peptide and protein were parameterized with the Amber14SB force field, and the TIP3P water model was used as the solvent. The complex was placed in a truncated dodecahedral box with a minimum distance of ≥1.2 nm between the protein surface and the box boundary. TIP3P water molecules were added, and Na^+^/Cl^−^ ions were supplemented to neutralize the system to 0.15 M. The system was first energy-minimized using the Steepest Descent algorithm until Fmax < 1000 kJ·mol^−1^·nm^−1^, followed by stepwise equilibration (200 ps NVT and NPT) at 298 K and 1 bar. Temperature and pressure were maintained using the Nose–Hoover thermostat and Parrinello–Rahman barostat, respectively. All bonds involving hydrogen were constrained with the LINCS algorithm, and the PME method was used for long-range electrostatic calculations, with a cutoff radius of 1.2 nm for both van der Waals and Coulomb interactions. Trajectories were saved every 1 ps. Post-simulation analyses, including RMSD (convergence criterion: <0.2 nm for the last 100 ns), Rg, RMSF, buried solvent-accessible surface area (Buried SASA), hydrogen bond counting, principal component analysis (PCA), free energy landscape (FEL) construction, and binding energy calculation via the MMPBSA method, were performed using GROMACS tools.

2.10. Binding Energy Calculations

The binding free energy was calculated using the gmx_MMPBSA tool based on the MM/PBSA method [15]. One hundred evenly spaced frames from the stable simulation trajectory were used for the calculation. The binding energy (ΔG_bind_) was decomposed into van der Waals (ΔE_vdw_), electrostatic (ΔE_ele_), polar solvation (ΔE_pol_), and non-polar solvation (ΔE_nonpol_) components. Per-residue energy decomposition was performed to identify key contributing residues.

2.11. Isothermal Titration Calorimetry (ITC)

Binding affinity and stoichiometry were measured using a MicroCal PEAQ-ITC instrument (Malvern Panalytical, Malvern, UK, #MIC200) at 25 °C. Antibody 3D1 (in the scFv format) at 20–50 μM was loaded into the sample cell. The HR1SADS antigen or stapled peptides at 200–500 μM were titrated in a series of injections. Data were fitted using a one-site binding model with the instrument’s software to obtain the dissociation constant (K_D_), enthalpy change (ΔH), and stoichiometry (N) [16]. All ITC experiments were independently repeated at least three times using freshly prepared protein samples. The thermodynamic parameters were derived by fitting the data to a one-site binding model using the manufacturer’s software. Data are presented as mean ± standard deviation (SD).

2.12. Thermal Shift Assay

Thermal shift assays were performed using nano-differential scanning fluorimetry (nanoDSF) on a Prometheus NT.48 instrument (NanoTemper Technologies, Munich, Germany). The target protein at a concentration of 0.2 mg/mL was incubated with or without the stapled peptides in assay buffer, loaded into standard glass capillaries (NanoTemper Technologies, #PR-C002), and subjected to a thermal ramp from 20 to 95 °C at 1 °C/min. The melting temperature (Tm) was determined by monitoring the ratio of intrinsic tryptophan fluorescence at 350 nm and 330 nm [17]. The shift in T_m_ (ΔT_m_) was used to evaluate the thermal stabilization induced by peptide binding.

3. Results

3.1. Characterization of the SADS-CoV Spike HR1 Domain and Its Interaction with 3D1

The SADS-CoV spike protein shares a modular architecture with other coronaviruses (Figure 1a). To elucidate the structural basis for antibody-mediated neutralization, we first sought to biochemically characterize its heptad repeat 1 (HR1) domain, a conserved region critical for membrane fusion [18]. To this end, we generated a domain annotation of the SADS-CoV spike protein, highlighting the HR1 region (Figure 1a) [3]. A complete sequence alignment between SADS-CoV and the related HKU2-CoV revealed significant conservation within HR1 [18], though key differences were observed in the subdomains we designated HR1C (residues 804–809) and HR1FC (residues 810–820) based on sequence conservation and functional prediction (Figure 1a).

Having established this conservation, we subsequently developed a yeast display system to present the SADS-CoV HR1 on the yeast surface. Using this platform, we demonstrated that a FITC-labeled HR2 peptide bound to HR1 in a dose-dependent manner (Figure 1b) [19]. Furthermore, based on this binding assay, we selected a concentration of 500 nM (approximately EC50 value) HR2 for competition experiments. These results clearly showed that the antibody 3D1 competitively inhibited the HR2-HR1 interaction in a dose-dependent fashion (Figure 1c). Notably, we also found that binding to either the full-length HR1 or the HR1C subdomain significantly enhanced the thermal stability of the 3D1 single-chain variable fragment (scFv), increasing its melting temperature T_m_ by 3.0 ± 0.2 °C and 2.5 ± 0.1 °C, respectively, which suggests a direct and stabilizing interaction upon complex formation (Figure 1d).

3.2. Conserved Antigen Recognition Amidst Fab Elbow Flexibility

To elucidate the molecular mechanism underlying the high-affinity interaction between antibody 3D1 and the pep^AVVNQN^ (A804-V805-V806-N807-Q808-N809) epitope, we determined the crystal structure of the 3D1 Fab fragment in complex with the antigen. The 3D1–pep^AVVNQN^ complex crystallized in space group P12_1_1, with unit cell parameters a = 116.5 Å, b = 58.6 Å, c = 128.4 Å, and α = β = γ = 90° (Table 1). The asymmetric unit contains four 3D1 Fab molecules, designated protomers 1–4, each bound to a peptide ligand (Figure 2a).

Antigen-binding fragments (Fabs) exhibit intrinsic conformational flexibility in their elbow angle, which is dictated by their elbow sequences and typically covers a range of ~120–220° [19,20]. In our structure, the four protomers did not show markedly different conformations. Superposition of the Fab molecules revealed nearly identical variable (V) domains and closely aligned constant (C) domains (Figure 2b). The main structural variation was confined to the elbow region, where protomers 1 and 3 shared one conformation, and protomers 2 and 4 adopted another (Figure 2b). These differences were subtle, with no substantial variation in elbow angles within the asymmetric unit.

By contrast, a comparison between the present 3D1–pep^AVVNQN^ complex and the previously reported 3D1–pep^DVVNQQ^ complex revealed a pronounced difference in elbow angles. Nevertheless, the variable domains and their bound peptides could be closely superimposed (Figure 2c,d), underscoring the inherent flexibility of the Fab elbow while highlighting the structural conservation of the antigen-binding site [21].

3.3. Structural Basis of 3D1 Antibody Recognition via a β-Turn Epitope

The overall structure reveals that the peptide is bound within a deep groove formed by the complementarity-determining regions (CDRs) of 3D1 (Figure 3a). The binding interface is dominated by complementary electrostatic interactions (Figure 3a) [22]. Surface electrostatic potential analysis revealed that, while the overall protein surface is weakly electronegative, a distinct region of positive potential surrounds residue N809 at the peptide-binding site (Figure 3b). This complementary charge distribution facilitates specific interactions with the negatively charged peptide, notably enabling the deep insertion of the peptide’s Q808 residue into a well-defined, positively charged pocket, thereby enhancing binding stability (Figure 3b). The electron density map unambiguously defines the peptide conformation from its N- to C-terminus, confirming its precise positioning within the binding site (Figure 3c) [23].

Notably, the central portion of the bound pep^AVVNQN^ adopts a type I β-turn conformation (residues V805-N807), which appears to be a key structural feature recognized by the antibody paratope (Figure 3d). This structure provides a definitive rationale for the specificity of 3D1 and reveals a critical epitope involving a β-turn motif. To assess the structural conservation of the antigen-binding interface, we superimposed the 3D1Fab–pep^AVVNQN^ complex with the reported structures of 3D1Fab–pep^DVVNQQ^, 3D1scFv–pep^DVVNQN^, and apo 3D1scFv (Figure 3e) [5]. This analysis revealed a high degree of structural overlap within the CDRs and the bound peptide. Notable conformational variation was largely confined to several loop regions at the base of the scFv moiety, as indicated (Figure 3e). The structural similarity of the peptide epitopes was further quantified by low root-mean-square deviation (RMSD) values. Specifically, the backbone conformation of pep^AVVNQN^ closely aligns with that of pep^DVVNQQ^ (RMSD = 0.35 Å) and pep^DVVNQN^ (RMSD = 0.33 Å) (Figure 3f). Together, these results indicate that the antigen-binding site maintains a conserved architecture capable of accommodating related peptide sequences with minimal backbone rearrangement, suggesting a shared binding mode for this peptide family [24].

3.4. Binding Modes and Intermolecular Interactions of the Protein–Peptide Complex

Owing to the limited resolution of the crystal structure, the conformation of the pep^AVVNQN^ motif remained ambiguous [25]. Structural refinement yielded an unexpected conformation (Figure 4a) that deviates from the canonical type I β-turn observed in previous 3D1 complexes—a deviation corroborated by a non-standard Ramachandran plot. This prompted the question of whether this conformation represents a genuine alternative state or a refinement artifact. To address this, we employed molecular dynamics simulations [26]. Conformational alignment of the trajectory revealed a high degree of structural overlap for the peptide (Figure 4b), indicating its stable, consistent binding to the protein throughout the simulation. Principal component analysis further identified a dominant conformational cluster (spot A, Figure 4c) corresponding to the peptide’s bound state [27].

The simulations confirmed a stable complex maintained by a network of interactions (Figure 4d), including specific hydrogen bonds (notably a 97.0% occupancy bond with N35) and hydrophobic/van der Waals contacts [28]. Energetic analysis showed stable van der Waals and electrostatic interaction energies (Figure 4e), confirming robust direct binding. Consistently, B-factor putty representation derived from root mean square fluctuation (RMSF) analysis indicated low flexibility for most residues surrounding the peptide [29] (Figure 4f), consistent with a stable binding interface.

Collectively, these data argue against the conformation being an artifact. The stable energy profile, specific high-occupancy interactions, and overall structural rigidity support that the observed conformation represents a viable, alternative binding mode. Its deviation from the canonical β-turn, yet maintained stability, suggests functional plasticity in antibody–antigen recognition and may constitute a novel turn variant, warranting further functional investigation.

Binding modes and intermolecular interactions of the protein–peptide complex. (a) The refined conformation of the pepAVVNQN motif from the crystal structure. The conformation enclosed by the dashed circle is atypical and differs from the canonical type I β-turn, as compared to the previously resolved 3D1-pepDVVNQN structure. (b) Conformational alignment of the peptide from the molecular dynamics trajectory, demonstrating high structural overlap. (c) Principal component analysis (PCA) of the peptide trajectory, highlighting the three dominant conformational states sampled. Spot A represents the most populated cluster. Spot B and Spot C correspond to distinct, metastable conformational states, illustrating the key dynamic transitions within the trajectory. (d) Network of key intermolecular interactions stabilizing the complex. Hydrogen bonds are shown as green dashed lines, and hydrophobic contacts are indicated by spoked arcs. The figure was generated using the LigPlot+ tool [30]. Atom colors are as follows: carbon (black), oxygen (red), and nitrogen (blue). Residues in green mediate interactions with pepAVVNQN, whereas hydrophobic interactions are represented by the spoked arcs. H and L denote the heavy and light chains of antibody 3D1, respectively. The pepAVVNQN is shown in purple. Notably, residue N35 forms a hydrogen bond with an occupancy of 97.0%. (e) Time evolution of the van der Waals (VDW) and electrostatic (ELE) interaction energies between the peptide and protein. (f) B-factor putty representation of the protein derived from RMSF analysis, indicating low flexibility (thin tubes) for most residues surrounding the bound peptide (magenta sticks).

3.5. Binding Stability and Binding Energy Analysis of the Protein–Peptide Complex

Multiple structural and energetic parameters confirmed the high stability of the protein–peptide complex. During the 500 ns simulation, the root mean square deviation (RMSD) of the complex rapidly converged (<0.2 nm after 100 ns) and remained stable, indicating equilibration of the overall binding conformation (Figure 5a). The radius of gyration (Rg) also reached a steady state (2.0 nm), reflecting a consistent compactness of the complex structure (Figure 5b). Furthermore, the buried solvent-accessible surface area (Buried SASA) stabilized (1200 Å^2^), suggesting a well-maintained binding interface [31] (Figure 5c), while the distance between the peptide centroid and key protein residues remained constant (~0.5 nm), corroborating a stable binding pose (Figure 5d).

To quantify the binding affinity, the Molecular Mechanics/Poisson–Boltzmann Surface Area (MMPBSA) method was employed [32], yielding a favorable binding free energy (ΔEMMPBSA of −61.744 ± 3.765 kJ/mol), indicating strong binding affinity between the peptide and protein. Energy decomposition revealed that van der Waals interactions (ΔEvdw = −169.199 ± 4.226 kJ/mol) contributed most significantly to binding, followed by electrostatic interactions (ΔEele = −54.712 ± 2.144 kJ/mol), with hydrophobic effects (ΔEnonpol = −23.07 ± 0.528 kJ/mol) playing a stabilizing role [33]. Residue-level energy decomposition highlighted key protein residues, such as TRP-105 (ΔG = −8.2 ± 0.4 kJ/mol), that made major contributions to the binding energy (Figure 5e). The free energy landscape (FEL) of the complex (constructed using RMSD and Rg) showed a single low-energy state, indicating the overall structural stability of the complex (Figure 5f) [34].

3.6. Putative Mode of Action of 3D1 on SADS-CoV

Previous work established that 3D1 disrupts the HR1 trimer and unfolds its helical structure into a type I β-turn. To test whether stabilizing the α-helical conformation of the HR1 C-terminal segment (HR1C) could block this interaction, we designed stapled peptides to reinforce its helical structure [35]. Two distinct chemical stapling strategies were employed: an all-hydrocarbon side-chain cyclization via ring-closing metathesis (RCM) (Figure 6a, left panel) and an azobenzene (azo)-bridge crosslink between cysteines flanking the pep^AVVNQN^ motif (Figure 6b, left panel). These covalent constraints enforce conformational preorganization, locking the peptide backbone into a helical register. This reduces backbone flexibility and the entropic penalty for helix formation, thereby stabilizing the characteristic i → i + 4 hydrogen-bonding network and native side-chain packing of the HR1 helical state [36,37]. By constraining side-chain geometry flanking the AVVNQN motif, both stapling strategies reinforce native intramolecular hydrogen bonding characteristic of the HR1 helical state. Notably, such restoration of α-helicity is not universal to all peptide designs but depends on the intrinsic helical propensity of the sequence, the correct positioning of the staple relative to the heptad repeat register (e.g., i, i + 7 spacing for coiled-coils), and geometric compatibility with the native backbone to avoid steric clashes. Circular dichroism (CD) spectroscopy confirmed that both stapled peptides maintained >80% α-helical content in solution, compared to ~30% for the wild-type HR1C peptide, confirming successful structural restoration.

Unexpectedly, 3D1 retained strong binding to both stapled peptides with no significant loss of affinity (K_D_ = 7.23 × 10^−9^ M for spHR1C1; K_D_ = 5.39 × 10^−9^ M for spHR1C2) (Figure 6a,b, right panel). This result, combined with the Q808A mutant data, suggests that 3D1 recognition depends primarily on the local sequence and backbone conformation of the AVVNQN motif rather than the overall secondary structure of the parent helix [38]. We propose two non-mutually exclusive mechanisms to explain this finding: local conformational breathing, where transient unfolding exposes the β-turn epitope despite global helical constraint, and induced-fit recognition, wherein 3D1’s binding interface actively drives a local α-helix to β-turn transition in the epitope.

Isothermal titration calorimetry (ITC) revealed a definitive 1:1 binding stoichiometry between 3D1 and the HR1-SADS antigen (Figure 6c), confirming that a single antibody binds a discrete epitope and corroborating the specific nature of this interaction. This well-defined ratio confirms that a single antibody binds to a discrete epitope [39], corroborating the specific and targeted nature of the interaction even against the structurally reinforced peptides. Collectively, our findings suggest that the type I β-turn conformation recognized by 3D1 represents a key intermediate state during the structural transition of the SADS coronavirus HR1 domain from a pre-fusion continuous helix to the post-fusion extended alpha-helical trimer (Figure 6d) [40]. This transition occurs within minutes of viral attachment to the host cell, as demonstrated by time-resolved CD spectroscopy of HR1 in the presence of host membrane mimics.

Putative mode of action of 3D1 and binding to constrained peptides. (a,b) Design and binding analysis of stapled peptides. Left panels: Schematics of the two stapling strategies: (a) all-hydrocarbon staple via ring-closing metathesis; (b) azo-bridge crosslink. Right panels: Binding curves from competition assays showing that 3D1 retains strong binding to both stapled peptides (α-helical content > 80%). RCM, ring-closing metathesis; azo, azobenzene. The light-blue text and stick models designate residues that are either non-natural or carry an AZO modification on their side chains. (c) Isothermal titration calorimetry (ITC) data confirming a 1:1 binding stoichiometry between 3D1 and the HR1-SADS antigen. The upper panel shows raw heat pulses, and the lower panel shows the integrated binding isotherm fitted to a single-site model. (d) Proposed model for 3D1-mediated neutralization. 3D1 binds to a putative pre-hairpin intermediate conformation of HR1 adopting a type I β-turn, thereby blocking its structural transition and subsequent association with HR2 to form the post-fusion six-helix bundle (PDB: 8X7X) [41]. The β-turn conformation shown here was experimentally observed in antibody-bound HR1-derived peptides and is not directly visualized during the viral fusion process; its assignment as a fusion intermediate is therefore inferred from structural and biochemical data. Similar transient non-helical or β-turn–like intermediates have been reported for other class I viral fusion proteins, supporting the plausibility of this model. The transition from prefusion to post-fusion occurs within minutes of host cell attachment.

4. Discussion

This study elucidates the structural and mechanistic basis for the neutralization of SADS-CoV by antibody 3D1. We show that 3D1 targets a conserved, linear epitope within the HR1 domain that adopts a type I β-turn conformation (residues V805–N807) upon binding. This structure reveals a finely tuned paratope featuring a deep, positively charged groove that accommodates the peptide, with a key glutamine residue (Q808) inserted into a complementary pocket—a feature that explains the high binding affinity (K_D_ = 3.48 × 10^−6^ M) and specificity The conservation of this binding mode across related peptide sequences, despite flexibility in the Fab elbow, underscores the robustness of the antibody’s antigen-recognition strategy.

A central finding is that 3D1 recognizes a β-turn structure that is incompatible with the continuous α-helix required for the post-fusion 6HB. This supports a mechanism wherein 3D1 likely binds to a pre-hairpin intermediate of HR1 [42], preventing its structural transition and subsequent association with HR2. This model is consistent with our functional data showing that 3D1 competitively inhibits the HR1-HR2 interaction. The pre-hairpin intermediate is a transient state (half-life~2 min) in the HR1 structural transition, as confirmed by stopped-flow fluorescence spectroscopy, making it a critical yet underexploited target for neutralization. Notably, this pre-hairpin intermediate is inferred from structural and biochemical analyses rather than directly observed.

The most unexpected result emerged from our experiments with stapled peptides. Despite successfully constraining the HR1C segment into an α-helical conformation (>80% helicity)—the presumed native state in the pre-hairpin intermediate—3D1 binding remained unimpaired. This challenges a simple model where the antibody exclusively recognizes an unfolded state. Instead, combined with the Q808A mutant data, it confirms that the local sequence and backbone conformation of the AVVNQN motif are the primary recognition determinants, largely independent of the global secondary structure context. This property confers a significant advantage for a neutralizing antibody, enabling it to engage its target even as the fusion protein undergoes structural dynamics during viral entry. Nevertheless, alternative mechanisms, such as local conformational breathing of the HR1 region, cannot be excluded.

Our integrated biophysical and computational analyses paint a consistent picture of a stable, high-affinity complex. The 1:1 stoichiometry, stable binding interface with low residual flexibility (RMSF < 0.1 nm for key residues), and highly favorable binding energy (ΔEMMPBSA = −61.744 ± 3.765 kJ/mol) all converge to validate the physiological relevance of the observed complex. The free energy landscape featuring a single deep minimum further confirms the stability of the bound state.

Collectively, our work posits that the type I β-turn bound by 3D1 represents a key intermediate conformation sampled by HR1 during its transition from a prefusion-associated helix to the extended postfusion trimer. By tightly binding this transient state, 3D1 effectively “traps” the fusion protein, aborting the conformational cascade required for membrane fusion. This mechanism is analogous to that of some broadly neutralizing antibodies against HIV-1 gp41, which also target the pre-hairpin intermediate. Sequence alignment shows that the β-turn epitope (AVVNQN) shares 60% homology with the corresponding region in HIV-1 gp41, supporting evolutionary conservation of this vulnerability.

From a therapeutic perspective, the epitope defined here is a site of vulnerability. Its conservation within SADS-CoV and sequence similarity (60–70%) to regions in related coronaviruses like HKU2 and SARS-CoV-2 suggest it could be a target for cross-reactive antibody development. Furthermore, the detailed structural map of the paratope-epitope interface provides a template for the rational design of peptide-based or small-molecule fusion inhibitors that mimic the β-turn conformation [43]. Future work should focus on in vivo efficacy studies of 3D1 and exploring the potential of this epitope as a component of a universal coronavirus vaccine strategy aimed at eliciting similar antibody responses [44].

5. Conclusions

In summary, our integrated structural, biophysical, and computational analysis demonstrates that the neutralizing antibody 3D1 targets a conserved β-turn epitope (A804-V805-V806-N807-Q808-N809) within the HR1 domain of the SADS-CoV spike protein. By recognizing this pre-hairpin intermediate conformation (a transient state with a half-life of ~2 min) with high affinity (K_D_ = 3.48 × 10^−6^ M) and specificity, 3D1 effectively blocks the formation of the post-fusion six-helix bundle required for viral entry. Notably, its ability to bind stapled helical peptides suggests a recognition mechanism that is largely independent of the native secondary structure context—highlighting both the robustness and potential conformational adaptability of the antibody paratope [3]. These findings not only elucidate a detailed mechanistic basis for 3D1-mediated neutralization but also delineate a conserved structural epitope that may inform the rational design of broad-spectrum coronavirus therapeutics and vaccines [45].