Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies

Abstract

A subset of bovine antibodies have an exceptionally long third heavy-chain complementarity determining region (CDR H3) that is highly variable in sequence and includes multiple cysteines. These long CDR H3s (up to 69 residues) fold into a long stalk atop which sits a knob domain that is located far from the antibody surface. Three new bovine Fab crystal structures have been determined to decipher the conserved and variable features of ultralong CDR H3s that lead to diversity in antigen recognition. Despite high sequence variability, the stalks adopt a conserved β-ribbon structure, while the knob regions share a conserved β-sheet that serves as a scaffold for two connecting loops of variable length and conformation, as well as one conserved disulfide. Variation in patterns and connectivity of the remaining disulfides contribute to the knob structural diversity. The unusual architecture of these ultralong bovine CDR H3s for generating diversity is unique in adaptive immune systems.

Fig. 1

Bovine ultralong CDR H3 sequence features and numbering scheme. Sequence alignment of bovine CDR H3 regions in this study with the germline-encoded V_HBUL, D_H2, and J_H1 gene segments. The highly abundant Tyr, Gly, Ser residues encoded in the D_H2 gene are colored in purple. The conserved canonical Cys^H92 (yellow) and Trp^H103 (cyan) that generally define the start and end of CDR H3 are indicated in all sequences. The length of each CDR and number of non-canonical Cys residues (also yellow) are shown on the far right. The numbering scheme (below the sequences in red) used here for bovine ultralong CDR H3s will follow Kabat numbering until the region encoded by D_H2 (shown in blue), which is easily identifiable by a highly conserved CPD motif with cysteine typically at position D2. This D region is numbered sequentially with a D identifier, from D1 to D50, then the region encoded by the J_H1 gene is numbered J1, J2, before returning to Kabat numbering at position H101 (two residues preceding the conserved Trp^H103 highlighted in cyan).

Fig. 2

Bovine Fab structures and sequence alignments. (A) Crystal structures of bovine Fabs E03, B11, BLV1H12 (PDB 4K3D), BLV5B8 (PDB 4K3E), and A01 in a schematic representation. The light chains are in pink and heavy chains in light blue. The core β-strands (as defined by DSSP) in the distal CDR H3 knob domains are colored yellow, green, and blue. (B) The bovine V_λ sequence (top) is aligned with the V_λ sequence from human antibody KOL (PDB 2FB4) (61). The five bovine V_H sequences (bottom) are aligned with the V_H sequence from human Fab PGT121 (PDB 4JY4) (24). The bovine H3 regions are aligned based on structural conservation, with the type I β-turn residues in red and β-strand residues colored as in (A).

Fig. 3

Architecture and relative disposition of bovine CDR H3 stalk and knob domains. (A) When the V_H domains of five bovine antibodies are superimposed (left), very little residual movement is seen in the stalk regions. CDR H3’s from bovine antibodies B11, BLV1H12, BLV5B8, A01, and E03 are shown in red, yellow, green, blue, and pink, respectively, with orthogonal views on left and right. (B) In contrast, when the knob domains are superimposed by their conserved type I β-turn and β-strand core, the difference in the position of the supporting stalks is much larger, showing that different knob domains have different relative orientations with respect to the conserved stalk and V_H core.

Fig. 4

Secondary and tertiary structure and topology of the bovine CDR H3 knob domain. The knob domain secondary and tertiary structure (A) and primary structure topology diagrams (B) are shown from left to right for bovine Fabs E03, B11, BLV1H12, BLV5B8, and A01. The spatially conserved disulfide from Cys^D2 to a Cys in the second β-strand in the knob domain is colored in red, and the other disulfides are color coded to match the topology diagrams. The β-strands 1, 2, and 3 are colored yellow, green and blue in all panels. Gly^D2 in A01 (that is usually a Cys in other bovine Fabs) is highlighted in red on a white background.

Fig. 5

Descending strand of the bovine CDR H3 stalk regions. Each Fab has conserved aromatic residues in the descending strand of the stalk that likely help stabilize the stalk β-ribbon. A kinked base region around H103 is found at the H3 base in all five bovine antibodies.

Fig. 6

Comparison of bovine CDR loop conformations with CDR canonical classes. (A) The H3 ‘kink’ or ‘bulge’ in 2FB4 (left) and B11 (right) are shown. In a ‘normal’ kinked base, Trp103 NE1 hydrogen bonds to a neighboring carbonyl as in 2FB4, but in the bovine CDR H3, Trp103 NE1 hydrogen bonds to the side chain of Thr^L46. (B) The CDR regions L1, L2, L3, H1 and H2 are shown superimposed with canonical reference CDRs from published structures. Bovine CDRs are shown in gray, and reference CDRs in red. Sequences for the CDRs are shown below the superpositions. The sequences for CDR H1 are identical in all five bovine structures, but differ for CDR H2, while the λ chain CDR sequences for all five bovine Fabs are identical. As the bovine λ chains are therefore also highly similar in structure, only one bovine CDR loop is shown in each of the superpositions. The L1 loops are very similar to canonical class 5λ/13a, except at Gly^L31 where a difference is observed in the position and orientation of its carbonyl oxygen. The L2 loops belong to class 1λ/7a. The L3 loops would be predicted to belong to class 5λ/11a, but the bovine loop tip bends away from the canonical structure to support the H3 stalk. The H1 loops for B11, A01, E03, and BLV5B8 (left) align fairly well with canonical class 1/10a, but both Fabs in BLV1H12 (center) have distorted H1 loops that interact with each other via crystal packing interactions. The H2 loops belong to class 1/9a.

Fig. 7

Structural homology of the bovine CDR H3 knob domain with the cyclotide Kalata B1. (A) The closest structural homologue to the knob domain, as identified with the PDBeFold server, is the small, cyclic peptide (cyclotide) Kalata B1. Superposition of the bovine Fab E03 knob domain (blue) with Kalata B1 (gray, PDB 1N1U) is shown. The E03 Cys^D2-D13 disulfide is shown in red at the same spatial location as the Kalata B1 disulfide. In the sequence alignment (below), the 16 underlined residues superimpose with an RMSD on Cα atoms of 1.6 Å. The red Cys residues in the sequence alignment represent the spatially conserved disulfide bond. (B) The E03 knob with core β-strands (yellow, green, and blue) and conserved disulfide (red). (C) Kalata B1 with homologous regions colored as for E03.