This study, through molecular dynamics simulations of 19 bovine antibodies, reveals the dynamic properties of ultralong CDRH3 and its interactions with light chains, providing a critical theoretical basis for understanding structural flexibility and functional optimization in bovine antibodies.
Literature Overview
The article 'Comparative Molecular Dynamics Study of Bovine Antibodies with Ultralong CDR H3', published in the journal Antibodies, reviews 19 bovine antibodies containing ultralong CDR H3 regions (ulCABs). Using molecular dynamics (MD) simulations, the research analyzed conformational stability, structural dynamics, and intermolecular interactions of ulCABs, revealing that their extended conformations in crystal structures are unstable in solution and exhibit significant dynamic variations. Additionally, the study observed stable interactions between the CDRH3 knob domain and light chains (LC) in approximately half of the systems, offering new insights into ulCAB structural stability.
Background Knowledge
ulCABs (bovine antibodies) feature ultralong CDRH3 regions where the knob domain is stabilized by disulfide bonds and connected to the antibody core via β-strands. In crystal structures, these stalk and knob domains typically adopt extended conformations, but crystal packing effects may constrain their dynamic behavior. This study employs MD simulations across 19 ulCAB systems to investigate dynamic properties under different environments, including conformational changes, sequence diversity, and LC interactions. It also explores how these dynamic features can guide mini-antibody design improvements for enhanced stability and binding affinity, providing theoretical support for antibody engineering.
Research Methods and Experiments
All 19 ulCAB structures were sourced from the Protein Data Bank. Structural alignment was performed to analyze sequence diversity, while missing flexible residues were reconstructed using the ModLoop server to ensure structural integrity. Each system underwent microsecond-scale MD simulations in explicit solvent with Amber 22 force field for energy minimization and system equilibration. Post-simulation analyses included RMSD calculations, inter-residue distance measurements, binding free energy analysis (MM/GBSA), and main chain dihedral angle fluctuation (TAF) analysis. Structural deviations, including rotational and translational motions of the knob domain, were further evaluated using cpptraj.
Key Conclusions and Perspectives
Research Significance and Prospects
The study elucidates structural flexibility and conformational dynamics of ulCABs, offering novel perspectives for understanding antibody functional evolution. Future work should validate stalk–knob conformational stabilization through designed variants, particularly in antigen recognition mechanisms. These findings also establish structural foundations for engineering antibody fragments with improved stability for antiviral and oncology applications.
Conclusion
Through large-scale MD simulations, this study systematically characterized the structural dynamic features of CDRH3 in ulCABs. Results demonstrate that while CDRH3 adopts extended conformations in crystal structures, significant flexibility emerges in solution with pronounced system-specific variations. Stable knob–LC interactions observed in ~50% of systems suggest critical roles in maintaining functional conformations. These insights provide essential guidance for ulCAB structural modeling and antibody fragment engineering, highlighting potential strategies such as conformation-stabilizing mutations or optimized cleavage site design to enhance mini-antibody stability and binding efficiency. The data also establishes structural frameworks and design principles for antigen-targeted therapeutic antibody development.
