<br/>The long-believed paradigm of static canonical structures determining binding properties and specificity of antibodies has recently shifted, based on our studies. We revealed that one single static structure is not sufficient to capture the high flexibility of all CDR loops, in particular of the CDR-H3 loop, and thus the paratope should be described as conformational ensemble in solution. In contrast to this prevalent static view of the binding interface, we demonstrate a dynamic perspective not only of the paratope, but of whole Fvs and Fabs. We show that antibodies exist as ensembles of paratope states. These paratope states are defined by a characteristic combination of CDR loop conformations and interdomain orientations, which interconvert into each other in the micro-to-millisecond timescale by correlated loop and interdomain rearrangements. Thus, to fully understand the antibody binding interface and to elucidate the molecular mechanism of antibody-antigen binding, the strongly correlated CDR loop conformations and the interfaces should be characterized as ensembles in solution.<br/>This dynamic view of the antibody binding site is particularly important to identify dominant conformations in solution when the available X-ray structure is distorted by crystal packing effects or if no experimental structure has been determined. In various examples, we have observed for the CDR-H3 loop and for the whole antibody paratope that the dominant ensemble in solution coincides with the binding competent conformation, which is not necessarily the case with the (apo) X-ray structure.<br/> <br/>Consequently, to predict antibody-complexes with docking, the high flexibility of the paratope by considering multiple CDR loop conformations, often referred to as ensemble docking or flexible docking, must be taken into consideration. However, incorporating multiple paratope conformations only increases accuracy when all important ensembles in solution are sampled and thermodynamically weighted by their respective ensemble probabilities. Otherwise, the inclusion of distinct paratope conformations without thermodynamic or kinetic information might introduce more inaccuracies and noise to the docking results. Thus, to benefit from paratope ensembles in solution, both structural and thermodynamic information is critical, as without such thermodynamic weighting, high energy conformations could dominate the docking results and consequently deteriorate the docking success.

Antibody structure prediction and docking improved by paratope ensembles in solution

ACS Spring 2022

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