V_H‐V_L interdomain dynamics observed by computer simulations and NMR

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 Statement Antibodies 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.

Surprisingly Fast Interface and Elbow Angle Dynamics of Antigen-Binding Fragments

“…This high variability in the V H -V L interdomain distribution has been reported for different IL-1β antibody fragments in agreement with the respective NMR ensembles (Fernández-Quintero et al, 2020c). By applying fast Fourier transformation to the interface angles, timescales of 0.1-10 GHz could be assigned to the fastest collective interdomain movements (Fernández-Quintero et al, 2020c). With the increasing number of available Fab X-ray structures, it was noted that these fragments also display a high variability in the elbow angle, which is defined as the angle between the pseudo-two-fold axes relating V H to V L and C H 1 to C L (Sotriffer et al, 2000;Stanfield et al, 2006).…”

“…ABangle is a computational tool (Dunbar et al, 2013;Bujotzek et al, 2015Bujotzek et al, , 2016Fernández-Quintero et al, 2020c) used to characterize the relative orientations between the antibody variable domains (V H and V L ) using six measurements (five angles and a distance). A plane is projected on each of the two variable domains.…”

“…The high variability in the V H -V L interdomain orientation is an additional feature of antibodies, which directly increases the size of the antibody repertoire (Chothia et al, 1985;Vargas-Madrazo and Paz-García, 2003;Bujotzek et al, 2016;Knapp et al, 2017;Fernández-Quintero et al, 2020c). This high variability in the V H -V L interdomain distribution has been reported for different IL-1β antibody fragments in agreement with the respective NMR ensembles (Fernández-Quintero et al, 2020c). By applying fast Fourier transformation to the interface angles, timescales of 0.1-10 GHz could be assigned to the fastest collective interdomain movements (Fernández-Quintero et al, 2020c).…”

T-Cell Receptor CDR3 Loop Conformations in Solution Shift the Relative Vα-Vβ Domain Distributions

“…Previous studies have shown that the relative interdomain orientations reveal a high variability and fluctuate in the 0.1 GHz timescale. These results suggest that short molecular dynamics simulations are sufficient to capture the majority of accessible interdomain orientations ( 73 ). In agreement with these studies we observe that the slow component of the relative Vα-Vβ dynamics strongly correlates with conformational CDR3 loop rearrangements, which occur on the micro-to-millisecond timescale.…”

Antibodies exhibit multiple paratope states influencing VH–VL domain orientations

“…For all CDR loops conformational transitions between canonical clusters and additional dominant solution structures have been observed 18,27,28 . Additionally, these conformational transitions between different CDR loop states occur in the micro-tomillisecond timescale 18,27,28,43 , while the relative V H and V L interdomain dynamics can be captured in the 0.1-10 ns timescale 44,45 .…”