# Microsecond Dynamics of Fc–CD16a Recognition: Impact of Mutations, Core Fucosylation, and Fc Asymmetry

## TL;DR

This study uses simulations to explore how mutations and fucose affect antibody interactions with immune cells, guiding better antibody design.

## Contribution

The study reveals how specific mutations and fucose modifications affect Fc-CD16a interactions at atomic resolution.

## Key findings

- Only S239D and H268F consistently stabilize the CD16a interface.
- Fucosylation reduces binding stability by disrupting glycan-mediated contacts.
- An asymmetric Fc variant maintains high-affinity binding through novel hydrophobic and electrostatic interactions.

## Abstract

Background/Objectives: Antibody-dependent cellular cytotoxicity relies on the interaction between the Fc region of immunoglobulin G1 (IgG1) and the CD16a receptor. While removal of core fucosylation on Fc and introduction of the DFTE mutation set (S239D, H268F, S324T, I332E) are known to enhance CD16a binding, the detailed contributions of these engineered sites in solution remain incompletely defined. Methods: Here, we employed 1 µs molecular dynamics simulations to map, at atomic resolution, the interaction networks stabilizing pre-formed Fc-CD16a complexes, including afucosylated Fc-wild-type, DFTE-engineered, Fc-fucosylated, and asymmetrically engineered Fc variants. Results: Our results show that only S239D, present on both Fc chains, and H268F on chain A consistently contribute to stabilizing the CD16a interface, while I332E does not form persistent interactions. Glycan–protein contacts are primarily intrachain, with transient interchain glycan–glycan interactions not contributing significantly to complex stability. Fucosylation on Fc significantly reduces binding stability by disrupting peripheral interactions and critical glycan-mediated contacts. Notably, the asymmetric Fc variant, in which the two heavy chains carry distinct sets of substitutions, retains high-affinity binding despite lacking S239D and carrying core fucose, through a novel hydrophobic cluster and reinforced peripheral electrostatic interactions. Conclusions: Altogether, these findings provide a quantitative framework for how targeted mutations and fucose modifications remodel Fc-CD16a interactions, offering insights for the rational design of next-generation therapeutic antibodies.