Hiding in plain sight: structure and sequence analysis reveals the importance of the antibody DE loop for antibody-antigen binding

Simon Kelow, Roland Dunbrack, Jared Adolf-Bryfogle

Abstract:

Antibody variable domains contain “complementarity-determining regions” (CDRs), the loops that form
the antigen binding site. CDRs1-3 are recognized as the canonical CDRs. However, a fourth loop sits
adjacent to CDR1 and CDR2 and joins the D and E strands on the antibody v-type fold. This “DE loop” is
usually treated as a framework region, even though mutations in the loop affect the conformation of the
CDRs and residues in the DE loop occasionally contact antigen. We analyzed the length, structure, and
sequence features of all DE loops in the Protein Data Bank (PDB), as well as millions of sequences from HIV1 infected and naïve patients. We refer to the DE loop as H4 and L4 in the heavy and light chains,
respectively. Clustering the backbone conformations of the most common length of L4 (6 residues)
reveals four conformations: two κ-only clusters, one λ-only cluster, and one mixed κ/λ cluster. Most H4
loops are length-8 and exist primarily in one conformation; a secondary conformation represents a small
fraction of H4-8 structures. H4 sequence variability exceeds that of the antibody framework in naïve
human high-throughput sequences, and both L4 and H4 sequence variability from λ and heavy germline
sequences exceed that of germline framework regions. Finally, we identified dozens of structures in the
PDB with insertions in the DE loop, all related to broadly neutralizing HIV-1 antibodies (bNabs), as well as
antibody sequences from high-throughput sequencing studies of HIV-infected individuals, illuminating
a possible role in humoral immunity to HIV-1.