Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution

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.

Germline-Dependent Antibody Paratope States and Pairing Specific VH-VL Interface Dynamics

“…These backbone rearrangements in the paratope occur in the micro-tomillisecond timescale. unique interactions stabilizing the V H -V L interface (33,73). Astonishingly, we observe the highest variations in the relative V H -V L distributions for the least stable Fab.…”

“…Figures 3 – 6 depict paratope states in solution of different germline pairings and do not only describe shifts in the V H -V L interface distributions, but also reveal germline pairing specific interface orientations. These characteristic V H -V L orientations are defined by CDR loop backbone and sidechain rearrangements, which result in unique interactions stabilizing the V H -V L interface ( 33 , 73 ). Astonishingly, we observe the highest variations in the relative V H -V L distributions for the least stable Fab.…”

“…In particular, residue 71 H [Kabat nomenclature ( 80 )], has been shown to co-determine the canonical conformation of the CDR-H2 loop, according to whether there is a bulky residue or a small side-chain present and thus bringing the CDR-H1 and CDR-H2 loops closer to each other ( 27 , 30 , 81 ). Especially interesting is that the 71 H residue is part of the Vernier-zone residues, which have been reported to play a critical role in the humanization process and for rational design of antibodies in general as they can influence antibody specificity and affinity ( 73 , 79 , 82 , 83 ). Differences in these framework residues might contribute to the distinct backbone and sidechain dynamics observed in the 5I15 and 5I19.…”

Ensembles in solution as a new paradigm for antibody structure prediction and design

“…11,[13][14][15][16][17][18] Furthermore, the different amino acids at position H71 (Kabat nomenclature) 10,12 are thought to influence both the position and the canonical cluster assignment of the CDR-H2 loop, and thus potentially affect antigen binding. 17,19,20 Generally, the major determinants of specificity and affinity of these five CDR loops for an antigen are the size, shape and biophysical complementarity of their surface residues and their relative positions to each other. 11 The CDR-H3 loop reveals the highest diversity in length, sequence and structure and has the ability to adopt various different conformations during the V(D)J recombination and somatic hypermutation.…”

How Communication Pathways Bridge Local and Global Conformations in an IgG4 Antibody: a Molecular Dynamics Study

“…18 Molecular dynamics (MD) simulations, on the other hand, offer a valuable tool for the investigation of the interplay between stabilizing interactions, fluctuation correlations, and conformational variability at different levels of resolution and experimental conditions. [19][20][21][22] In silico structural investigation of immunoglobulins and antigenantibody complexes has been successfully employed with different objectives, which include the detailed description of the dynamics of CDR loops and the transitions between their conformational states, [23][24][25] as well as the comprehension of structural rearrangements and allosteric modifications following antigen binding. [26][27][28] Here, we employ atomistic MD simulations to investigate the internal dynamics of fulllength pembrolizumab, a humanized IgG4 antibody used in immunotherapy, whose full structure has been experimentally solved 29 (Figure 1).…”