Abstract
Antibody variable domains contain “complementarity determining regions” (CDRs), which are solvent exposed loops that form the antigen binding site. Three such loops, CDR1, CDR2, and CDR3, are recognized as the canonical CDRs. However, there exists a fourth solvent-exposed loop, the DE loop, adjacent to CDR1 and CDR2 that joins the D and E strands on the antibody v-type fold. The DE loop is usually treated as a framework region, and as such, structural and genetic studies of antibodies often ignore this loop; yet, their lengths, structures, and sequences are variable and they contact the antigen in some antigen-antibody complex structures. We analyzed all of the structures and sequences of DE loops, which we refer to as H4 and L4 in the heavy and light chain variable domains respectively, as well as searched through millions of antibody sequences from both HIV-1 infected and naïve patients to look for human DE loop sequences with interesting features. Clustering the backbone conformations of the most common length of L4 (6 residues) reveals four dominant conformations, two of which contain only κ light chains, one of which contains only λ light chains, and one of which contains both κ and λ light chains. H4 loops in mammalian germlines are all of length 8 and their structures exist in only one conformational cluster. Length-8 L4 CDRs from a subset of λ5/λ6 germlines all have a backbone conformation very similar to that of the H4 length 8 cluster. Our structural classification of the DE loop uncovers its influence on CDR1 and CDR2 conformations, which in turn affect antibody binding. Furthermore, we show that H4 sequence variability exceeds that of the antibody framework in somatically mutated sequences from naïve human high-throughput sequences, and both L4 and H4 sequence variability from λ and heavy germline sequences also exceed that of germline framework regions. Finally, we identified a variety of insertions in DE loops present in dozens of structures of broadly neutralizing HIV antibodies in the PDB, as well as antibody sequences from high-throughput sequencing studies of HIV-infected individuals, thus illuminating a possible role in humoral immunity to HIV-1.