Abstract
The relative orientation of the two variable domains, VH and VL, influences the shape of the antigen binding site, that is, the paratope, and is essential to understand antigen specificity. ABangle characterizes the VH‐VL orientation by using five angles and a distance and compares it to other known structures. Molecular dynamics simulations of antibody variable domains (Fvs) reveal fluctuations in the relative domain orientations. The observed dynamics between these domains are confirmed by NMR experiments on a single‐chain variable fragment antibody (scFv) in complex with IL‐1β and an antigen‐binding fragment (Fab). The variability of these relative domain orientations can be interpreted as a structural feature of antibodies, which increases the antibody repertoire significantly and can enlarge the number of possible binding partners substantially. The movements of the VH and VL domains are well sampled with molecular dynamics simulations and are in agreement with the NMR ensemble. Fast Fourier transformation of the ABangle metrics allows to assign timescales of 0.1‐10 GHz to the fastest collective interdomain movements. The results clearly show the necessity of dynamics to understand and characterize the favorable orientations of the VH and VL domains implying a considerable binding interface flexibility and reveal in all antibody fragments (Fab, scFv, and Fv) very similar VH‐VL interdomain variations comparable to the distributions observed for known X‐ray structures of antibodies.Significance StatementAntibodies have become key players as therapeutic agents. The binding ability of antibodies is determined by the antigen‐binding fragment (Fab), in particular the variable fragment region (Fv). Antigen‐binding is mediated by the complementarity‐determining regions consisting of six loops, each three of the heavy and light chain variable domain VH and VL. The relative orientation of the VH and VL domains influences the shape of the antigen‐binding site and is a major objective in antibody design. In agreement with NMR experiments and molecular dynamics simulations, we show a considerable binding site flexibility in the low nanosecond timescale. Thus we suggest that this flexibility and its implications for binding and specificity should be considered when designing and optimizing therapeutic antibodies.
Highlights
Antibodies have become an important tool in therapeutics and clinical diagnostics.[1,2] This increasing relevance has motivated the development of computational techniques to study antibody structure and function.[3,4] The ability of antibodies to recognize a broad variety of pathogenic molecules is determined by the antigen-binding fragment (Fab), in particular the variable fragment region (Fv)
These high variations are obvious in comparison to the distributions of angles and the distance of the originally published 352 crystal structures found in the Protein Data Bank (PDB) displayed in the background
This timescale is orders of magnitude faster than that of the loop dynamics, especially of the complementarity-determining regions (CDRs)-H3 loop, which occur on the microsecond to millisecond timescales.[11]
Summary
Antibodies have become an important tool in therapeutics and clinical diagnostics.[1,2] This increasing relevance has motivated the development of computational techniques to study antibody structure and function.[3,4] The ability of antibodies to recognize a broad variety of pathogenic molecules is determined by the antigen-binding fragment (Fab), in particular the variable fragment region (Fv). Various studies only consider the Fv fragment to describe and investigate antigen-binding This reduces the system size and thereby decreases the computational time and costs.[5] The Fv fragment is the focal point of recombination and hypermutation events.[6,7,8,9,10,11] Antigen-binding is mediated by six loops of variable sequence and length denoted as the complementarity-determining regions (CDRs) which are distributed evenly over the heavy and light chain variable domains, VH and VL. Transitions between different states in this preexisting conformational space can occur on different timescales, and characterization of the thermodynamics and kinetics is vital to understand their conformational diversity.[17]
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