Abstract
Characterizing and understanding the antibody binding interface have become a pre-requisite for rational antibody design and engineering. The antigen-binding site is formed by six hypervariable loops, known as the complementarity determining regions (CDRs) and by the relative interdomain orientation (VH–VL). Antibody CDR loops with a certain sequence have been thought to be limited to a single static canonical conformation determining their binding properties. However, it has been shown that antibodies exist as ensembles of multiple paratope states, which are defined by a characteristic combination of CDR loop conformations and interdomain orientations. In this study, we thermodynamically and kinetically characterize the prominent role of residue 71H (Chothia nomenclature), which does not only codetermine the canonical conformation of the CDR-H2 loop but also results in changes in conformational diversity and population shifts of the CDR-H1 and CDR-H3 loop. As all CDR loop movements are correlated, conformational rearrangements of the heavy chain CDR loops also induce conformational changes in the CDR-L1, CDR-L2, and CDR-L3 loop. These overall conformational changes of the CDR loops also influence the interface angle distributions, consequentially leading to different paratope states in solution. Thus, the type of residue of 71H, either an alanine or an arginine, not only influences the CDR-H2 loop ensembles, but co-determines the paratope states in solution. Characterization of the functional consequences of mutations of residue 71H on the paratope states and interface orientations has broad implications in the field of antibody engineering.
Highlights
The rise of antibodies as important biotherapeutic proteins has sparked the interest in characterizing antibody structures and investigating structure–function relationships [1,2,3]
To facilitate the structure prediction of antibodies, five of these six complementarity determining regions (CDRs) loops have been assigned to so-called canonical clusters, assuming that they can only adopt a limited number of backbone conformations [5, 8,9,10,11]
We use a well-established protocol combining enhanced sampling techniques and classical molecular dynamics to investigate the influence of residue 71H on the whole paratope, the individual CDR loop dynamics and the respective relative VH–VL orientations
Summary
The rise of antibodies as important biotherapeutic proteins has sparked the interest in characterizing antibody structures and investigating structure–function relationships [1,2,3]. The variable fragment (Fv) exhibits the highest diversity of an antibody, as it is the focal point of somatic hypermutation and recombination events [6, 7]. This high diversity of the Fv is concentrated on six hypervariable loops, known as the complementarity determining regions (CDRs), which form the antigen binding site, the paratope. To facilitate the structure prediction of antibodies, five of these six CDR loops have been assigned to so-called canonical clusters, assuming that they can only adopt a limited number of backbone conformations [5, 8,9,10,11]. Within the obtained CDR loop ensembles in solution, transitions between the majority of canonical clusters and additional dominant solution structures were observed
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