CDR-H3 loop ensemble in solution – conformational selection upon antibody binding

Abstract

We analyzed pairs of protein-binding, peptide-binding and hapten-binding antibodies crystallized as complex and in the absence of the antigen with and without conformational differences upon binding in the complementarity-determining region (CDR)-H3 loop. Here, we introduce a molecular dynamicsbased approach to capture a diverse conformational ensemble of the CDR-H3 loop in solution. The results clearly indicate that the inherently flexible CDR-H3 loop indeed needs to be characterized as a conformational ensemble. The conformational changes of the CDR-H3 loop in all antibodies investigated follow the paradigm of conformation selection, because we observe the experimentally determined binding competent conformation without the presence of the antigen within the ensemble of pre-existing conformational states in solution before binding. We also demonstrate for several examples that the conformation observed in the antibody crystal structure without antigen present is actually selected to bind the carboxyterminal tail region of the antigen-binding fragment (Fab). Thus, special care must be taken when characterizing antibody CDR-H3 loops by Fab X-ray structures, and the possibility that pre-existing conformations are present should always be considered.

VH‐VL interdomain dynamics observed by computer simulations and NMR

“…Combining experimental NOE data with molecular dynamics simulations reveals in this example the same fast interdomain dynamics in the low nanosecond timescale for the different fragments analyzed. The timescale of interdomain reorientation is orders of magnitudes faster than the loop dynamics reflecting a hydrophobic interface with rather unspecific interactions that can be easily broken and result in low kinetic barriers, whereas loop dynamics is dominated by reorganization of hydrogen bond networks and electrostatic interactions characterized by strong forces and high barriers leading to much longer transition time scales . The distributions of interdomain orientations resulting from the time evolutions are very similar to distributions observed for antibody fragments in the PDB.…”

Antibodies exhibit multiple paratope states influencing VH–VL domain orientations

“…We observe strong correlations between the different CDR loop conformations. structure belongs to the dominant minimum in solution 28 . Also the relative interdomain orientation V H -V L differs 2°between the two available X-ray structures and both interface orientations are present within the obtained ensemble in solution.…”

T-Cell Receptor Variable β Domains Rigidify During Affinity Maturation

“…A previously published method characterizing the CDR-H3 loop ensemble upon antigen binding in solution 12,22 was used to investigate the conformational diversity of CDR 2 and HV 4 loops of TCR Vß variants in different stages of affinity maturation. Experimental structure information was available for all stages of affinity maturation and we used all available crystal structures as starting structures for molecular dynamics simulations.…”

“…Promiscuity might arise from a multitude of weakly populated conformations, each of which is able to bind different binding partners. Rigidification shifts the probability toward a small number of states and thereby reduces the amount of possible binding partners 20,[22][23][24] .…”