This study systematically reveals the dynamic effects of Fc region mutations, glycosylation modifications, and chain asymmetry on the CD16a binding interface through microsecond-scale molecular dynamics simulations, providing an atomistic mechanistic framework for antibody engineering.
Literature Overview
The article "Microsecond Dynamics of Fc–CD16a Recognition: Impact of Mutations, Core Fucosylation, and Fc Asymmetry", published in the journal "Antibodies", reviews and summarizes the dynamic network of interactions between the IgG1 Fc region and the CD16a receptor, with a focus on the effects of the DFTE mutation, core fucosylation, and Fc chain asymmetry engineering on binding stability. The study employs microsecond-scale molecular dynamics simulations to resolve non-covalent interactions among protein–protein, protein–glycan, and glycan–glycan at the atomic level, revealing dynamic changes in the binding interface under different modification conditions. Results indicate that the S239D and H268F mutations significantly enhance interface stability, whereas core fucosylation weakens binding by disrupting peripheral contacts. Notably, asymmetric Fc engineering, despite lacking S239D and retaining fucose, maintains high affinity through novel hydrophobic clusters and electrostatic networks. This study provides a dynamic perspective and theoretical basis for optimizing therapeutic antibody design.Background Knowledge
Antibody-dependent cellular cytotoxicity (ADCC) is a critical mechanism by which the immune system eliminates target cells, relying on the binding of the IgG antibody’s Fc region to the CD16a (FcγRIIIa) receptor on natural killer (NK) cells. CD16a belongs to the FcγR family, and its interaction with IgG Fc activates NK cells to release cytotoxic granules, killing tumor or infected cells marked by antibodies. Enhancing ADCC has become a key strategy in therapeutic antibody engineering. The Fc region of IgG1 binds CD16a through its CH2 domain, with binding affinity modulated by multiple factors, among which core fucosylation is particularly crucial: removal of the core fucose from the Fc N-glycan significantly enhances CD16a affinity, a strategy already applied in clinical therapeutic antibodies such as obinutuzumab. Additionally, affinity can be further increased through site-directed mutations (e.g., DFTE: S239D/H268F/S324T/I332E). However, the dynamic contributions of these modifications in solution remain incompletely understood, as static structures fail to capture conformational changes and interaction persistence. Recently, asymmetric Fc engineering has emerged, aiming to achieve receptor selectivity or signal modulation by introducing different mutations into the two heavy chains, although its molecular mechanisms require deeper elucidation. Therefore, analyzing the Fc–CD16a recognition mechanism from a dynamic perspective is essential for the rational design of next-generation therapeutic antibodies with higher affinity and selectivity.
Research Methods and Experiments
The study employed molecular dynamics (MD) simulations to perform 1-microsecond simulations on four pre-formed Fc–CD16a complexes, with four replicates per system, yielding a total of 16 microseconds of trajectory data. The four systems were: wild-type afucosylated Fc (Fc-af), DFTE quadruple-mutant afucosylated Fc (Fc4m-af), DFTE quadruple-mutant fucosylated Fc (Fc4m-f), and asymmetric-engineered fucosylated Fc (FcAs-f). All systems were built based on high-resolution crystal structures (PDB 3AY4, 3WN5), using homology modeling via CHARMM-GUI and MODELLER, and explicit-solvent, fully glycosylated simulations were conducted in GROMACS using the CHARMM36m force field. Trajectory analysis focused on the final 900 ns, with hydrogen bonds, ionic interactions, and van der Waals contacts quantified using MDAnalysis, RING 2.0, and custom scripts. Residue-level binding energies were evaluated using the MM/GBSA method, and system stability was verified using metrics such as RMSD, RMSF, radius of gyration, and SASA.Key Conclusions and Perspectives
Research Significance and Prospects
This study provides a dynamic map of the Fc–CD16a recognition process through microsecond-scale molecular dynamics simulations, going beyond the static view offered by traditional crystal structures. The findings not only explain the mechanisms of established engineering strategies (e.g., defucosylation, DFTE mutations) but also reveal novel molecular bases for how asymmetric engineering maintains high affinity, offering atomic-level guidance for rational antibody design. For example, future efforts could optimize H268 or peripheral hydrophobic regions, or design new asymmetric mutation combinations to balance affinity and selectivity.
Moreover, the study emphasizes the indirect role of glycan dynamics in Fc receptor recognition, suggesting that glycan homogeneity and protein sequence design should be jointly considered in antibody development. Although this study focuses on CD16a, its methods and framework can be extended to other FcγRs or FcRn, advancing the design of multi-receptor modulating antibodies. Future work could integrate free energy calculations or enhanced sampling methods to quantitatively predict mutation effects, or simulate the complex formation process to gain a comprehensive understanding of binding kinetics.
Conclusion
This study systematically analyzed the dynamic network of Fc–CD16a interactions through large-scale molecular dynamics simulations, revealing the fine-tuned regulatory mechanisms by which mutations, glycosylation, and chain asymmetry affect the binding interface. It was found that among the DFTE mutations, only S239D and H268F significantly contribute to interface stability, while I332E has limited impact; core fucosylation weakens binding by disrupting peripheral contacts, supporting its use as a strategy to enhance ADCC; most notably, asymmetric Fc engineering compensates for the absence of S239D and the presence of fucose through novel hydrophobic and electrostatic networks, maintaining high affinity. These findings not only deepen our understanding of Fc–receptor recognition mechanisms but also provide a dynamic, atomistically resolved theoretical framework for the rational design of next-generation therapeutic antibodies, aiding in the development of more effective and specific immunotherapies. This work demonstrates the power of long-timescale simulations in resolving the dynamic behaviors of macromolecular complexes, setting a new research paradigm in the field of antibody engineering.
