Introduction: Shark-derived single-domain antibodies (VNARs) exhibit unique structural adaptability, but the dynamic consequences of mutations remain poorly characterized. This computational study investigates how targeted mutations alter the conformational landscape of a VNAR, focusing on hypervariable (HVs), complementary-determining regions (CDRs), and framework (FW) residues critical for stability and function.

Methods: Conformational ensembles for the wild-type and mutant VNARs (with 13 mutations) in apo and complex lysozymes were generated using the ClustENMD unbiased sampling method, which integrates elastic network modeling, clustering, and molecular dynamics simulation. The ensembles were analyzed using Principal Component Analysis (PCA) to identify dominant motions; Linear Discriminant Analysis (LDA) and Random Forest were used to classify state-specific conformations and pinpoint residue-level discriminators. Centrality analyses of residue interaction networks were also applied to quantify changes in the VNAR allosteric communication. Additionally, the Essential Site Scanning Analysis (ESSA) was employed to identify potential allosteric sites on VNARs.

Results: PCA revealed divergent global dynamics, with the mutant exhibiting decreased flexibility. LDA identified several critical residues in the HV2, HV4, CDR1, CDR3, and FW3 that differ between the wild-type and mutant forms of VNAR, suggesting long-range allosteric effects. Central residues unique to the wild-type and mutant were extracted separately as a result of the centrality analyses, highlighting several residues in the five regions mentioned earlier, some of which overlap with those identified in the LDA findings. The Random Forest results also highlight the importance of HV4, FW3, and CDR3, in line with our ESSA results.

Conclusion: The mutations perturb VNAR dynamics predominantly in HV4, FW3, and CDR3 regions essential for antigen binding and structural integrity. These computational analyses not only decipher mutation-induced dynamical shifts, but also provide a blueprint for the rational design of stabilized VNARs. Our findings underscore FW3 as a potential allosteric regulator, offering new targets for engineering shark antibodies with enhanced therapeutic properties.