Large‐scale analysis of somatic hypermutations in antibodies reveals which structural regions, positions and amino acids are modified to improve affinity

Abstract

The principles of affinity maturation of antibodies (Abs), which underlies B cell-mediated immunity, are still under debate. It is unclear whether the antigen (Ag) binding site is a preferred target for mutations, and what the role of activation-induced deaminase (AID) hotspots is in this process. Here we report a structural analysis of 3495 residues that have been replaced through somatic hypermutations (SHMs) in 196 Abs. We show that there is no correlation between the propensity of an amino acid to be in AID hotspot and the probability that it is replaced during the SHM process. Although AID hotspots may be necessary to enable SHMs, they are not a major driving force in determining which residues are mutated. We identified Ab positions that are highly mutated and significantly affect binding. The effect of mutation on binding energy is a major factor in determining which structural regions of the Ab are mutated. There is a clear preference for mutations at the Ag-binding site. However, positions outside this region that also affect binding are often preferred targets for SHMs. As for amino acid preferences, a general trend during SHM is to make Ab-Ag interfaces more similar to protein-protein interfaces in general. In different regions of the Ab, there are different sets of preferences for amino acid substitution. This mapping improves our understanding of Ab affinity maturation and may assist in Ab engineering.

Origins of specificity and affinity in antibody–protein interactions

“…Short-chain hydrophilic side chains are particular suitable for this binding role because of the smaller side chain conformational entropy penalty by fixing the side chains in the interfaces. These notions are consistent with the studies showing that Tyr, Trp, and Ser are highly overrepresented in germ-line contacting residues (36), such that antibody repertoires derived from recombination of germ-line antibody sequences are highly functional in antigen recognition; somatic hypermutations reduce the population of these residue types where these residues are not involved in antibody-antigen interactions. Watermediated hydrogen bonding could contribute also to the affinity and specificity, but the magnitude could be relatively insignificant.…”

OptMAVEn – A New Framework for the de novo Design of Antibody Variable Region Models Targeting Specific Antigen Epitopes

“…An apparent trend is that charged residues are favorable in the affinity mature sequences while aromatic residues are disfavored. Despite the importance of aromatic residues in the binding to antigens [31], this finding is in agreement with previous studies that tyrosine and tryptophan are abundant in the germline genes and the degree of aromaticity is typically reduced during affinity maturation [58]. Meanwhile, the number of polar residues is slightly increased.…”

Understanding differences between synthetic and natural antibodies can help improve antibody engineering

“…To build a large non-redundant set of natural Abs, a previously published non-redundant dataset of 196 Ab-Ag complexes 61 was further filtered to create the current study data set of natural Ab-Ag complexes. The "CDRs Analyzer" cannot analyze single-chain variable fragment (scFv) Abs, Abs that contain disorder residues in the CDRs or non-standard amino acids, complexes that were solved by NMR and complexes composed of more than 25000 atoms.…”