Figures & data
Figure 1. Position of the DE loop in antibody structures
(a) V-type fold according to Bork et. al. The DE loop and CDRs are indicated. Strand A forms beta-strand pairing interactions with both strand B and strand G. (b) Example of antibody Fab fragment (light chain in green, heavy chain in blue). (c) Top-down view of antibody combining site. The canonical CDRs and the DE loop are marked in panel C and are represented in the same colors in panel B.
Table 1. Map between various numbering schemes within H4 and L4 loops
Figure 2. Ramachandran plots for part of the D strand, the DE loop, and part of the E strand (IMGT residues 77–90) for the most common DE loop lengths
(a). ϕ (x-axis) and ψ (y-axis) for residues in length-8 H4 loops, and the 3 anchor residues before and after the loop. IMGT residue numbers are provided at the bottom of each panel. (b). ϕ,ψ plots for residues in length-6 L4 loops, and the 3 anchor residues before and after the loop
Table 2. DE loop canonical families
Figure 3. Canonical conformations of L4 and H4
ϕ,ψ plots for each residue in the DE loop for each of the L4 and H4 DE loop clusters from the full data set (no EDIA cutoff) with their respective sequence logos. (a). L4 length-6 loops. (b). L4 length-8 loops. C. H4 length-6 loops. D. H4 length-7 loops. E. H4 length-8 loops
Figure 4. Structures of all DE loop germline-length clusters
(a) The antibody light-chain DE loop (L4-6) backbone is shown for each cluster where L4-6-1 (PDB 3d9aL, blue) sits closest to the antibody domain, L4-6-3 (1mjuL, green) hinges slightly away and flips the second carbonyl of the DE loop backbone about 180° relative to the other clusters, L4-6-2 (4unuA, magenta) hinges further away from the domain than L4-6-1 and L4-6-3, and L4-6-4 (6frjH, orange) sits the furthest away from the domain. The stems of the DE loop are colored dark gray. (b). Same representation as in (A), but for the sole L4-8-1 (5jpjA, green) cluster. (c). Same representation as in (A), but for the H4-8-1 (5e7bA, cyan) and H4-8-2 (6bliJ, orange) clusters(d). Same representation as in (A), but for the H4-6-1 cluster (6i9iH, green). (e). Same representation as in (A) but for the H4-7-1 cluster (6dbdD).
Figure 5. Comparison of H4 and L4 clusters with structural homology
(a). Superposition of several high-resolution heavy chain H4-8-1 structures (cyan, PDB chains: 2x1qA, 4qyoB, 2vxvH), and L4-8-1 structures (green, PDB chains: 1cd0A, 2w01A, 3h0tA) aligned by the stem of CDR4 (colored in gray) show structural similarity between the two clusters. B. Same superposition as in 5A, but for L4-6-3 (green, 1mujL, 6qnkC, 6mv5L) and H4-6-1 (cyan, 6i8iH, 6cf2A, 6banH) clusters.
Table 3. Co-occurrence of L1/L4 pairs from structures in the PDB
Figure 6. Various characteristic hydrogen bonds between the DE loop and CDR1. Hydrogen bonds are labeled CDR4.resnumAtom/CDRn-cluster.resnumAtom (e.g. H4.6O/H1-13-1.2N). If a hydrogen bond is specific to a particular cluster, that is included in the nomenclature
(a). Side-chain/backbone hydrogen bond L4.Tyr6OH/L1-11-2.8N (yellow/red) common in L1-11-2, (b). Side-chain/backbone hydrogen bond H4.Arg1NH1/H2-10-1.3O (yellow/purple) common in H2-10-1. (c). Side-chain/backbone hydrogen bonds L4.3O/L1.6 N and L4.4O/L1.2 N (both yellow/red) common in L1-10, L1-11, L1-12, L1-13-1, L1-14-2, L1-15-1, L1-16-1, L1-17-1 clusters. (d). Backbone/backbone hydrogen bond L4.3N/L1-11-1.7O common in L1-11-1. (e). Backbone/backbone hydrogen bond L4-6-2.4O/L1-14-1.7 N common in L1-14-1. (f), Side-chain/backbone hydrogen bond H4.Arg1NH/H1-13.10O common in H1-13-1,2,3,4. (g). Side-chain/backbone hydrogen bond L4.R6NH/L1-12-3.7O and L4.R6NH/L1-12-3.9O common in L1-12-3. (h). backbone/side-chain hydrogen bonds L4.3 N/L1-15-1.S7OG and L4.3N/L1-15-1.D7OD common in L1-15-1. (i). L4.3 N/L1.N(−4)OD common in L1-16-1 and L1-17-1.
Figure 7. Sequence entropy in naïve human antibodies and human germlines
(a). Sequence entropy for 12 common germlines in a naive human antibody sample (>10,000 sequences for each germline). (b). Sequence entropy for human germlines derived from all IGKV, IGLV, and IGHV sequences from IMGT. From left to right, the pink shaded regions indicate CDR1, CDR2, and the DE loop. CDR3 is omitted due to varying lengths and different diversification mechanisms.
Table 4. Average sequence entropies for CDR and framework regions
Table 5. HIV-1 bNAbs with insertions in L4 and/or H4
Figure 8. Alignment of a subset of gp120 binding HIV-1 bNAbs representing all unique DE loop sequences
(a). Aligned structures of H4 inserted bNAbs of the VRC and 3BNC series (one structure per H4 sequence). The inset shows hydrophobic contacts between the antibody-antigen interface, as well as a salt bridge between the first Arg residue in H4 and the antigen. (b). Structure of the 35O22 antibody found in 35 PDB entries with an H4 of length 16. The inset shows a hydrophobic residue interaction with hydrophobic residues in the helix of the antigen. (c). Structure of the 8ANC195 antibody with an H4 of length 12 represented in 16 PDB entries (one structure per H4 sequence). The inset shows hydrophobic residues interacting with hydrophobic residues in a loop of the antigen. (d). Aligned structures of L4-inserted bnAbs binding to HIV-1 gp120 (one representative per unique L4 sequence of length 9). The inset shows hydrophobic contacts at the antibody-antigen interface, including hydrophobic contacts to glycosylation residues on gp120 (gray sticks). (e). Alignment of antibody 35O22 heavy chain DNA and protein sequences around the DE loop segment with the DNA and protein sequences of the germline IGHV1-18*02. The 35O22 DE loop protein sequence is at the bottom in yellow. The IGHV1-18*02 DE loop protein sequence is at top. The duplicated regions are underlined. The IGHV1-18*02 is identical in both duplicated regions, while the 35O22 sequence has diverged either prior to or after duplication or both. Identical base pairs or amino acids between 35O22 and IGHV1-18*02 are highlighted in gray. (f). Alignment of antibody PGT122 light chain DNA and protein sequences around the DE loop segment with the DNA and protein sequences of the germline IGLV3-21*01. The PGT122 DE loop protein sequence is at the bottom in yellow. The IGLV3-21*01 DE loop protein sequence is at top. The duplicated regions are underlined. The IGLV3-21*01 is identical in both duplicated regions, while the PGT122 sequence has diverged either prior to or after duplication or both. Identical base pairs or amino acids between PGT122 and IGLV3-21*01 are highlighted in gray.
Figure 9. Buried surface area for each CDR at the antibody-antigen interface of HIV-1 bNAbs that bind to gp120 in the PDB
(a). Buried surface area plot for 18 PDB structures (non-redundant by chain) with insertions in H4. (b). Buried surface area plot for 31 PDB structures (non-redundant by chain) with insertions in L4.
Figure 10. DE loop and DE loop adjacent insertions from a large antibody sequencing data set from HIV-infected individuals
(a). Insertions in κ gene antibodies. (b). Insertions in λ gene antibodies. C. Insertions in heavy gene antibodies.
Supplemental material