Conservation of the C-type lectin fold for massive sequence variation in a Treponema diversity-generating retroelement

Abstract

Anticipatory ligand binding through massive protein sequence variation is rare in biological systems, having been observed only in the vertebrate adaptive immune response and in a phage diversity-generating retroelement (DGR). Earlier work has demonstrated that the prototypical DGR variable protein, major tropism determinant (Mtd), meets the demands of anticipatory ligand binding by novel means through the C-type lectin (CLec) fold. However, because of the low sequence identity among DGR variable proteins, it has remained unclear whether the CLec fold is a general solution for DGRs. We have addressed this problem by determining the structure of a second DGR variable protein, TvpA, from the pathogenic oral spirochete Treponema denticola. Despite its weak sequence identity to Mtd (∼16%), TvpA was found to also have a CLec fold, with predicted variable residues exposed in a ligand-binding site. However, this site in TvpA was markedly more variable than the one in Mtd, reflecting the unprecedented approximate 10(20) potential variability of TvpA. In addition, similarity between TvpA and Mtd with formylglycine-generating enzymes was detected. These results provide strong evidence for the conservation of the formylglycine-generating enzyme-type CLec fold among DGRs as a means of accommodating massive sequence variation.

Fig. 1.

Structure of TvpA. (A) Two views of TvpA in ribbon representation (α-helices are gold, β-sheets blue, and loops are gray, except for the VR, which is entirely pink). (B) Two orientations of TvpA in ribbon representation with the β-flap (residues 52–94) in blue, insert 1 (residues 103–136) in magenta; insert 1′ (residues 153–195) in light orange; and insert 2 (residues 212–258) in green. Other portions of TvpA are in gray.

Fig. 2.

The FGE-type CLec fold of TvpA. Ribbon representation (Top; β-strands in blue and α-helices in red) and topology diagram (Bottom) of the CLec fold in (A) TvpA (2Y3C), (B) Mtd (1YU0), and (C) hFGE (2HI8). Disulfide bonds in hFGE are displayed as sticks. For clarity, inserts are ghosted, the β-flap of TvpA is not shown, and only the C-terminal domain of Mtd is displayed.

Fig. 3.

(A) Surface representation of TvpA, with the VR facing the viewer. Variable hydrophobic residues (A, V, L, I, Y) are green, variable hydrophilic residues (S, N, D, E, R, C) blue, and variable glycines pale orange. (B) VR of TvpA in ribbon representation. The main chain is in gray, side chains of variable residues are in red (red dots are variable glycines), and nonvariable aromatic residues are in orange. (C) Superposition of the VR of TvpA (blue) and Mtd-P1 (light orange) in Cα representation. The spheres represent the position of variable residues in each protein. (D) Stabilization of the VR (gray, variable residues indicated by red spheres) by insert 2 (green) in Cα representation. Dashed line indicates hydrogen bonds.

Fig. 4.

Relationship to FGE. Superposition of the VR of TvpA (blue) and the catalytic site of hFGE (green) in Cα representation. Bottom: Sequence alignment of the regions shown above. TvpA variable residues denoted by spheres and hFGE catalytic residues by arrows. Identical residues are in red, and chemically similar ones in blue.