Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor

Abstract

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.

Extending human IgG half-life using structure-guided design

“…Further, the 250-helix residues demonstrated enhanced deuterium exchange at low pH in the absence of FcRn, suggesting that a pH-specific conformational change occurred within this region. 23,24 …”

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity

“…33 Mass spectrometry coupled to HX (HX-MS), extends the HX technique to complex, multi-domain macromolecules like mAbs. [37][38][39][40][41][42][43] Recently, we described a novel HX-MS method to map protein interfaces formed between mAbs undergoing reversible protein-protein interactions directly at up to 60 g/L. 39 Here, we applied this technique to investigate the molecular mechanism by which an IgG1 mAb (mAb-J) undergoes reversible self-association, and further probed this mechanism by a variety of other biophysical techniques.…”

The Area Between Exchange Curves as a Measure of Conformational Differences in Hydrogen-Deuterium Exchange Mass Spectrometry Studies

“…In practice, recovery of individual exchange rate constants from HDX-MS data is rarely achieved due to the generally acknowledged ill-conditioned and under-determined characteristics of the problem, although many creative approaches toward extracting additional information have been developed [14–15]. …”