A New Clustering of Antibody CDR Loop Conformations

Abstract

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 colleagues, 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 using affinity propagation. With this data set of over 300 non-redundant antibody structures, we were able to cover 28 CDR-length combinations (e.g., L1 length 11, or “L1-11” in our 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; κ/λ) and one purely by the presence and 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 of them have multiple conformational clusters including ten for which Chothia had only one canonical class. We have a total of 72 clusters for the 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” vs. “non-bulged” conformations based on the presence or 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 the new classification will lead to improved methods for antibody structure prediction and design.

An Ultra-specific Avian Antibody to Phosphorylated Tau Protein Reveals a Unique Mechanism for Phosphoepitope Recognition

“…In contrast, chickens use a much longer V H CDR3 repertoire and frequently stabilize these long binding loops by introducing non-canonical disulfide bonds (32). In the structure presented here, a descriptive example of that mechanism is highlighted, along with a V CDR1 structure, which may be common in chicken antibodies but is distinctly different from any hitherto described structure in mammals (58,69).…”

“…We therefore systematically compared the CDR sequences and conformations of this clone with the canonical CDRs from humans and rodents as classified by North et al (58). CDR-H3 was not included in the analysis because it is known to have diverse lengths and structures (58). We found that chicken CDR-L2, CDR-L3, CDR-H1, and CDR-H2 all adhere to canonical conformations seen in mammalian structures and could be classified into clusters L2-8, L3-11, H1-13, and H2-10, respectively (58).…”

“…CDR-H3 was not included in the analysis because it is known to have diverse lengths and structures (58). We found that chicken CDR-L2, CDR-L3, CDR-H1, and CDR-H2 all adhere to canonical conformations seen in mammalian structures and could be classified into clusters L2-8, L3-11, H1-13, and H2-10, respectively (58). In contrast, chicken CDR-L1 has a conformation that is distinct from all canonical CDR-L1 clusters described in humans and rodents to date (Fig.…”

Overlapping hotspots in CDRs are critical sites for V region diversification

“…The black vertical lines are mutations in dC on either strand and are presumed to have been initiated by the direct action of AID, whereas the gray vertical lines are mutations in A:T residues and are presumed be a result of error-prone MMR (47). After recent precedents (1,30,48), the CDRs indicated by the horizontal gray bars at the top of the figure are based on the Kabat criteria (49) combined with analysis of the crystal structure of the IGHV3-23*01 V region, rather than the IMGT definition (48). It is obvious that the frequency of these independent and unique somatic mutations in Fig.…”

Conformation-Dependent Recognition of HIV gp120 by Designed Ankyrin Repeat Proteins Provides Access to Novel HIV Entry Inhibitors

“…Antibodies can do this, too, but they can also bind to an unstructured terminal peptide or long loop, which adapts to a pocket or groove between the antibody variable domains. Conversely, the complementary determining regions (CDRs) of the antibody, especially a long CDR-H3, can bind within a pocket of the target or at the side of a domain (69). The preference of DARPins for recognizing structural components could be advantageous in selecting HIV envelope-directed inhibitors, particularly as recent findings of potent broadly active neutralizing antibodies revealed a high prevalence of antibodies recognizing conformational inter-and intraprotomer loop-spanning binding domains (70,71).…”