Abstract
ABSTRACT In the past decade, the relevance of antibodies as therapeutics has increased substantially. Therefore, structural and functional characterization, in particular of the complementarity-determining regions (CDRs), is crucial to the design and engineering of antibodies with unique binding properties. Various studies have focused on classifying the CDR loops into a small set of main-chain conformations to facilitate antibody design by assuming that certain sequences can only adopt a limited number of conformations. Here, we present a kinetic classification of CDR loop structures as ensembles in solution. Using molecular dynamics simulations in combination with strong experimental structural information, we observe conformational transitions between canonical clusters and additional dominant solution structures in the micro-to-millisecond timescale for all CDR loops, independent of length and sequence composition. Besides identifying all relevant conformations in solution, our results revealed that various canonical cluster medians actually belong to the same kinetic minimum. Additionally, we reconstruct the kinetics and probabilities of the conformational transitions between canonical clusters, and thereby extend the model of static canonical structures to reveal a dynamic conformational ensemble in solution as a new paradigm in the field of antibody structure design. Abbreviations: CDR: Complementary-determining region; Fv: Antibody variable fragment; PCCA: Perron cluster analysis; tICA: Time-lagged independent component analysis; VH: Heavy chain variable region; VL: Light chain variable region
Highlights
The importance of characterizing and engineering the structure of antibodies to improve specificity, stability and suitability as biotherapeutics has increased substantially in the past decades.[1– 4] Natural occurring antibodies are symmetric Y-shaped proteins, and each symmetric unit consists of a heavy and light chain
SI Figure S1 illustrates an overlay of the canonical cluster median crystal structures, which lie in the same kinetic minimum with the obtained Complementary-determining region (CDR)-L1 loop ensemble in solution
We characterized the structural diversity of all CDR loops in solution and provide a kinetic and thermodynamic analysis of the conformational space supported by strong experimental structural information
Summary
The importance of characterizing and engineering the structure of antibodies to improve specificity, stability and suitability as biotherapeutics has increased substantially in the past decades.[1– 4] Natural occurring antibodies are symmetric Y-shaped proteins, and each symmetric unit consists of a heavy and light chain. Sequence and structural diversity of antibodies is concentrated on six hypervariable loops, known as complementaritydetermining regions (CDRs), located within each of the two antibody antigen-binding domains. Three hypervariable loops (CDR-H1, CDR-H2, CDR-H3 and CDR-L1, CDR-L2, CDR-L3) are located on the heavy and light chain, respectively.[5]. Various studies have focused on classifying five of the six CDR loops into canonical conformations, except the CDR-H3 loop, assuming that, depending on the length and sequence composition, antibody CDR loops only adopt a limited number of main-chain conformations.[6]. The highest variability in length, sequence, and structure can be observed for the CDR-H3 loop. The CDR-L3 loop reveals a comparable diversity to the CDR-H3 loop, but, without the contribution of a D gene, the degree of variability is lower.[9]
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