Affinity improvement of specific scFvs interacting with the Neisseria meningitidis factor H-binding protein: a molecular dynamics simulation approach and experimental study
Findings underscore affinity maturation as a powerful strategy for engineering high-affinity antibody fragments by integrating in silico maturation, molecular dynamics simulations, and experimental validation.
Abstract
In silico approaches are becoming increasingly important for enhancing the affinity of antibodies and antibody fragments, including single-chain variable fragments (scFvs). In this study, we present an in silico affinity-maturation workflow designed to improve the affinity of an scFv directed against
Neisseria meningitidis
factor H-binding protein (fHbp) by integrating in silico maturation, molecular dynamics (MD) simulations, and experimental validation. Approximately 300 single-point substitutions were designed at scFv residues predicted to interact with fHbp, and the resulting V3 (R99D) scFv–fHbp complexes were analyzed through all-atom MD simulations. Guided by bioinformatic analyses, the top V3 (R99D) variant was cloned and expressed, and its binding activity was validated by ELISA.
MD analyses identified five variants, Y35H, L31R, R99D, R99H, and A100K, that exhibited markedly enhanced complex stability. In all variants, the scFv secondary structure remained intact, whereas fHbp showed shrinkage relative to the scFv in the V3 (R99D)–fHbp complexes during the simulations. Hydrogen-bonding patterns and inter-residue distances further supported the improved affinity of the variants relative to the native form. The R99D variant showed the highest binding affinity, with an experimentally measured value of 67.93 × 10⁹ M⁻¹.
Collectively, these findings underscore affinity maturation as a powerful strategy for engineering high-affinity antibody fragments.
The broadly neutralizing antibody VRC34.01, isolated from donor N123, recognizes the N-terminal fusion peptide of the HIV-1 envelope glycoprotein gp41 and inhibits viral membrane fusion. A detailed molecular understanding of this interaction is essential for elucidating the basis of peptide recognition and guiding antibody optimization. Here, molecular dynamics (MD) simulations combined with free energy perturbation (FEP) calculations were used to systematically investigate the effects of point mutations on both the fusion peptide and the antibody. Mutational analyses of key peptide residues showed that most substitutions reduce binding affinity, indicating that the native peptide sequence is finely optimized for recognition by VRC34.01. Decomposition of binding free energies further reveals that these effects are primarily driven by changes in van der Waals interactions associated with hydrophobic packing at the interface. In contrast, in silico mutagenesis of antibody interfacial residues identifies a small subset of affinity-enhancing mutations, including A33I, L96I and S93T. Structural analyses indicate that these mutations improve binding by optimizing local hydrophobic packing without altering the overall binding mode. These results provide a quantitative and structural framework for understanding fusion peptide recognition and highlight a general strategy for antibody affinity optimization through subtle interfacial packing refinement.
Mei Feng, Nan Wang, Yi Song et al.· Journal of Physical Chemistr...· 0 citations
The ongoing Mpox (monkeypox) outbreak underscores the need for effective therapeutic antibodies. Antibody VACV-66 binds strongly to the vaccinia virus D8 protein but shows markedly reduced cross-reactivity to the MPXV homolog E8L, despite >95% sequence identity. In this study, we combined AlphaFold3 modeling, molecular dynamics simulations, and ASGB-IE free energy calculations to investigate this mechanism and rationally design high-affinity antibody mutations targeting E8L. Our calculations revealed that the single N18D mutation in E8L (where N18 in D8 corresponds to D18 in E8L) introduces electrostatic repulsion with antibody residue D34L, triggering local conformational changes that displace key residue R20 from the aromatic cage formed by W106H and Y33L. Based on this mechanism, saturation mutation of D34L identified three optimal mutations (D34LR, D34LK, and D34LN) that enhance binding free energy by removing electrostatic repulsion, restoring R20 insertion into the aromatic cage, and forming new favorable interactions. Quantitative analysis of the binding free energy contributions of residues N18 and R20 across different systems provided additional support for this mechanism. This work elucidates how a single viral mutation dampens antibody cross-reactivity and provides a general computational strategy for mechanism-guided antibody optimization against emerging orthopoxviruses. The identified D34LR, D34LK, and D34LN mutants represent promising candidates for experimental validation and therapeutic development.
Zhendong Li, Wenbin Qiu, Yanbiao Wang et al.· Physical Chemistry, Chemical...· 0 citations
Human metapneumovirus (HMPV) is a major cause of acute respiratory infections, especially among children and immunocompromised patients, but until now, no antiviral therapy has been approved. Nucleocapsid (N) protein, an ultra-conserved protein critical to the encapsidation of RNA and replication, is an attractive antiviral target. Herein, we used structure-based drug repurposing through the integration of high-throughput virtual screening, binding energy estimation by the merged molecular mechanics and generalized born/surface area (MM/GBSA) method, and long-timescale (1 μs) all-atom all-hydrogen atom molecular dynamics (MD) simulations to identify new HMPV N protein inhibitors. Of an initial set of 106 candidate compounds, two candidates, namely F0840-0186 and F3165-0800, emerged with the best docking scores (–6.81 and –6.99 kcal/mol) and MM/GBSA binding energies (–74.42 and –58.83 kcal/mol) upon the first round of screening. MD simulations validated the stability of these protein–ligand complexes with average root-mean-square deviation (RMSD) values of 2.5 and 2.8 Å to outperform the reference compound, namely ginkgolic acid (RMSD: 4.2 Å; ΔG_bind: –59.20 kcal/mol). Additional analysis on the structure stability and compactness of the complex included the free energy landscape and the principal component analysis. These computational results give predictive clues for potential HMPV inhibitors, for practical confirmation of the antiviral activity and drug potential.
M. Saeed, Gulam Rabbani, Mohamed Shantier et al.· Journal of King Saud Univers...· 0 citations
Structural findings suggest that contemporary mutations in the RBD may contribute to changes in receptor-binding interactions while preserving key structural features associated with host cell attachment in Influenza A(H3N2).
Karen M Hernandez-Gonzalez, A. S. Carranza-Aranda, J. Muñóz-Valle et al.· International Journal of Mol...· 0 citations
Combined genomic surveillance and structural analyses demonstrated that the investigated Omicron-associated mutations largely preserved the overall architecture of the M protein-Fab interaction interface while modulating residue-level energetic contributions and the conformational dynamics of the E protein.
Comparing wild-type XG83 and modified MuXG83 shows how allosteric tuning affects antibody-antigen compatibility in developing variations like Omicron, and indicates that non-epitope (potentially allosteric) changes to CDRH3 are also important while investigating potential development and neutralization before advancement.
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