Antibodies exhibit multiple paratope states that can differ in VH-VL domain orientations
Monica L. Fernández-Quintero, Alexander Bujotzek, Guy Georges, Klaus R. Liedl
Department of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Austria Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany
Abstract Paratope states in solution shift the relative VH-VL domain orientations 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 Antibodies exist as ensembles of partaope states, which are defined by a characteristic combination of CDR paratope states. These paratope states are defined by a characteristic combination of CDR loop loop conformations and interdomain orientations. conformations and interdomain orientations. They interconvert into each other in the micro-to- millisecond timescale by correlated loop and interdomain rearrangements. The relative V -V interdomain dynamics are surprisingly fast and occur in the 0.1 to 10 GHz timescale. We demonstrate that crystal packing effects can distort the paratope state and thus result in H L misleading X-ray structures. By advancing the repertoire of cutting-edge simulation techniques, for the first time we achieve a complete description of conformations, thermodynamics and kinetics of the whole binding paratope in solution.
These findings have broad implications in the field of antibody design and in the development of 7% 35% biotherapeutics as they provide a new paradigm in the understanding of CDR binding loop states, antibody-antigen recognition, relative VH and VL interface angles and elbow-angle distributions and their respective dynamics.
9% 23% CDR-H3 loop ensembles in solution – rigidification upon affinity maturation
The CDR-H3 loop, due to its high structural diversity, needs to be described as 18% 8% conformational ensemble in solution.
Affinity Maturation of a Naive anti-Lysozyme Antibody
This characteristic paratope states play a crucial role in the affinity maturation process and in the structural and functional characterization of the antibody binding site. Rigidification of the CDR-H3 Upon affinity maturation we observe a substantial Matured anti-Lysozyme Antibody loop conformational ensemble rigidification in both the CDR loop conformational diversity in solution as a consequence of and in the relative interdomain dynamics. affinity maturation.
Conclusion CDR loop ensembles in solution vs. canonical clusters from X-ray structures Antibody paratopes are flexible and exist in several different conformations. We characterize for several antibody fragments The CDR-L3 loop shows various conformational transitions between different canonical clusters kinetically accessible paratope states and observe correlated and should also be described as conformational ensembles in solution. binding interface loop rearrangements. Besides kinetic characterization of the paratope into different correlated CDR loop states, we also present a strong dependence of the CDR loop conformations on the relative interdomain orientation. We show that antibody CDR loop conformations are strongly correlated, reducing the combinatorics of possible states of the complete binding interface and shifting the relative VH and VL orientation and the elbow-angle distributions. These findings have broad implications in the field of antibody design and in the development of biotherapeutics as they provide a new paradigm in the understanding of CDR binding loop states,
antibody-antigen recognition and relative VH and VL interface angles and their respective dynamics.
References
• Fernández-Quintero, ML, et al. "Characterizing the Diversity of the CDR-H3 Loop Conformational Ensembles in Relationship to Antibody Binding Properties." Frontiers in immunology 9 (2018) • Fernández-Quintero, ML., et al. "CDR-H3 Loop Ensemble in Solution–Conformational Selection upon Antibody Binding." mAbs 11 (2019) • Fernández-Quintero, ML, et al. “Transitions of CDR-L3 Loop Canonical Cluster Conformations on the Micro- to-Millisecond Timescale.” Frontiers in immunology 10 (2019)
• Fernández-Quintero, ML, et al. “VH‐VL interdomain dynamics observed by computer simulations and NMR.” All CDR loops are highly flexible in solution and reveal transitions between different canonical Proteins 88 (2020) clusters. Thus, all CDR loops need to be described as conformational ensembles in solution. • Fernández-Quintero, ML, et al. “Antibody CDR loops as ensembles in solution vs. canonical clusters from X- ray structures.” mAbs 12 (2020) • Fernández-Quintero, ML, et al. “Antibodies exhibit multiple paratope states influencing VH-VL domain orientations.” Communications Biology (2020)
Acknowledgment
This project was supported by the Austrian Science Fund (FWF) via the grant [P30565] and [P30737].