Structural Basis of Antibody Conformation and Stability Modulation by Framework Somatic Hypermutation

Abstract

Accumulation of somatic hypermutation (SHM) is the primary mechanism to enhance the binding affinity of antibodies to antigens in vivo. However, the structural basis of the effects of many SHMs remains elusive. Here, we integrated atomistic molecular dynamics (MD) simulation and data mining to build a high-throughput structural bioinformatics pipeline to study the effects of individual and combination SHMs on antibody conformation, flexibility, stability, and affinity. By applying this pipeline, we characterized a common mechanism of modulation of heavy-light pairing orientation by frequent SHMs at framework positions 39H, 91H, 38L, and 87L through disruption of a conserved hydrogen-bond network. Q39LH alone and in combination with light chain framework 4 (FWR4L) insertions further modulated the elbow angle between variable and constant domains of many antibodies, resulting in improved binding affinity for a subset of anti-HIV-1 antibodies. Q39LH also alleviated aggregation induced by FWR4L insertion, suggesting remote epistasis between these SHMs. Altogether, this study provides tools and insights for understanding antibody affinity maturation and for engineering functionally improved antibodies.

Patterns of convergent somatic hypermutations in the adaptive immune response of Mus musculus

“…We identified 964 murine structures in the dataset created by Sheng et al which contains 3163 nonredundant, experimentally determined antibody structures from the SAbDab database. [3, 35] We then used IMGT’s DomainGapAlign tool to assign the most likely germline V gene for each antibody. [30]…”

“…[1, 2] Understanding the selection pressures in GC reactions that lead to this outcome and identifying patterns of SHMs has been a longstanding goal of immunological research. [3–5] Many SHMs are in or proximal to the binding site, where they add or optimize specific interactions with antigen. However, a substantial number of SHMs, referred to as framework mutations, are distal from the binding site, and it is often unclear if and how they improve affinity and/or specificity of the BCR.…”

“…[1,2] Understanding the selection pressures in GC reactions that lead to this outcome and identifying patterns of SHMs has been a longstanding goal of immunological research. [3][4][5] Many SHMs are in or proximal to the binding site, where they add or optimize specific interactions with antigen.…”

“…One hypothesis is that framework mutations are stabilizing the variable domain to compensate for destabilizing mutations in the binding site. [3,6] We and others have hypothesized that framework mutations rigidify the framework and thereby increase antigen-specificity. [7][8][9][10][11][12][13][14] It is also well known that the AID pathway that generates SHMs has strong biases for certain nucleotide motifs, and it has been suggested that some framework mutations are simply the result of neutral genetic drift.…”

“…[22][23][24][25][26][27][28] The majority of work to date regarding the mutability of antibody framework regions and their roles in structural evolution has been geared towards identifying key positions, residues, and relative orientations of heavy and light chain that modulate binding affinity in specific antibodies. [3,4,6,16,18,19,29] Perhaps the most comprehensive treatment of patterns of SHMs are the so-called gene-specific substitution profiles (GSSPs) developed by Sheng et al. [29] A GSSP shows the most frequent amino acid substitutions along a given germline V gene.…”