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Mechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design

ABSTRACT: Antibodies bind antigens via flexible loops called complementarity-determining regions (CDRs). These are usually 6-20 residues long. However, some bovine antibodies have ultra-long CDRs comprising more than 50 residues organized in a stalk and a disulfide-rich knob. The design features of this structural unit and its influence on antibody stability remained enigmatic. Here, we show that the stalk length is critical for the folding and stability of antibodies with an ultra-long CDR and that the disulfide bonds in the knob do not contribute to stability; they are important for organizing the antigen-binding knob structure. The bovine ultra-long CDR can be integrated into human antibody scaffolds. Furthermore, mini-domains from de novo design can be reformatted as ultra-long CDRs to create unique antibody-based proteins neutralizing SARS-CoV-2 and the Alpha variant of concern with high efficiency. Our findings reveal basic design principles of antibody structure and open new avenues for protein engineering. Certain bovine antibodies have ultra-long long complementarity-determining regions (CDRs) that contain a knob for antigen interaction, which is connected to the antibody through a stalk. Here, the authors combine biophysical experiments and MD simulations and show that the stalk length is critical for the folding and stability of these antibodies. The authors also demonstrate that ultra-long bovine CDRs can be grafted into human antibodies, and furthermore show that de novo designed mini-domains that bind to the SARS-CoV-2 spike protein with high affinity can be integrated as a knob in ultra-long CDRs in bovine and human antibodies, which neutralize SARS-CoV-2.

SUBMITTER: Svilenov H

PROVIDER: S-EPMC8602281 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Previous analyses of the complementarity-determining regions (CDRs) of antibodies have focused on a small number of "canonical" conformations for each loop. This is primarily the result of the work of Chothia and coworkers, most recently in 1997. Because of the widespread utility of antibodies, we have revisited the clustering of conformations of the six CDR loops with the much larger amount of structural information currently available. In this work, we were careful to use a high-quality data set by eliminating low-resolution structures and CDRs with high B-factors or high conformational energies. We used a distance function based on directional statistics and an effective clustering algorithm with affinity propagation. With this data set of over 300 nonredundant antibody structures, we were able to cover 28 CDR-length combinations (e.g., L1 length 11, or "L1-11" in our CDR-length nomenclature) for L1, L2, L3, H1, and H2. The Chothia analysis covered only 20 CDR-lengths. Only four of these had more than one conformational cluster, of which two could easily be distinguished by gene source (mouse/human; ?/?) and one could easily be distinguished purely by the presence and the positions of Pro residues (L3-9). Thus, using the Chothia analysis does not require the complicated set of "structure-determining residues" that is often assumed. Of our 28 CDR-lengths, 15 have multiple conformational clusters, including 10 for which the Chothia analysis had only one canonical class. We have a total of 72 clusters for non-H3 CDRs; approximately 85% of the non-H3 sequences can be assigned to a conformational cluster based on gene source and/or sequence. We found that earlier predictions of "bulged" versus "nonbulged" conformations based on the presence or the absence of anchor residues Arg/Lys94 and Asp101 of H3 have not held up, since all four combinations lead to a majority of conformations that are bulged. Thus, the earlier analyses have been significantly enhanced by the increased data. We believe that the new classification will lead to improved methods for antibody structure prediction and design.

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Mechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design

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