The Fab portion of immunoglobulin G has sites in the CL domain that interact with Fc gamma receptor IIIa

The interaction between IgG and Fc gamma receptor IIIa (Fc gamma RIIIa) is essential for mediating immune responses. Recent studies have shown that the antigen binding fragment (Fab) and Fc are involved in IgG-Fc gamma RIII interactions. Here, we conducted bio-layer interferometry (BLI) and isothermal titration calorimetry to measure the kinetic and thermodynamic parameters that define the role of Fab in forming the IgG-Fc gamma RIII complex using several marketed therapeutic antibodies. Moreover, hydrogen/deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS) were used to clarify the interaction sites and structural changes upon formation of these IgG-Fc gamma RIII complexes. The results showed that Fab in IgG facilitates the interaction via slower dissociation and a larger enthalpy gain. However, a larger entropy loss led to only a marginal change in the equilibrium dissociation constant. Combined HDX-MS and XL-MS analysis revealed that the CL domain of Fab in IgG was in close proximity to Fc gamma RIIIa, indicating that this domain specifically interacts with the extracellular membrane-distal domain (D1) and membrane-proximal domain (D2) of Fc gamma RIIIa. Together with previous studies, these results demonstrate that IgG-Fc gamma RIII interactions are predominantly mediated by the binding of Fc to D2, and the Fab-Fc gamma RIII interaction stabilizes complex formation. These interaction schemes were essentially fucosylation-independent, with Fc-D2 interactions enhanced by afucosylation and the contribution of Fab slightly reduced. Furthermore, the influence of antigen binding on IgG-Fc gamma RIII interactions was also investigated. Combined BLI and HDX-MS results indicate that structural alterations in Fab caused by antigen binding facilitate stabilization of IgG-Fc gamma RIII interactions. This report provides a comprehensive understanding of the interaction between IgG and Fc gamma RIII.

Automated summary

This study investigates the interaction between IgG and Fc gamma receptor IIIa (FcγRIIIa). The results show that the Fab of IgG facilitates the interaction by slowing dissociation and increasing enthalpy gain. The CL domain of Fab specifically interacts with the D1 and D2 domains of FcγRIIIa. Additionally, antigen binding plays a role in stabilizing the IgG-FcγRIIIa interactions.