Table 1.
Data collection and refinement statistics.
Fig 1.
(A) Sequence features of Tp0624. SP—signal peptide (approximately residues 1 to 60). Green bar—construct used for structural studies (T61-D476). Dashed box—region with predicted secondary structure elements but no sequence similarity to any characterized protein. Dotted box—putative OmpA-like C-terminal domain. (B) Orthogonal views of Tp0624 tertiary structure shown as a cartoon with beta-strands in green, alpha helices in purple, and connecting coil in grey. Note the slightly flattened, triangle-like architecture with two distinct sides defined by either beta-strands (left) or alpha-helices (right).
Fig 2.
Tp0624 is organized into two units consisting of D1-D2 and D3.
(A) Top: structure-based assignment of Tp0624 domains (D1, green; D2, cyan; D3, purple) and linkers (L1 and L2, yellow). Bottom: cartoon of Tp0624 tertiary structure colored by domain as in (top). (B) Table of inter-domain/linker interfaces of Tp0624 listed as “Interface Area in Å2 (# Hydrogen Bonds, # Salt Bridges)” as defined by PISA [37]. (C) Interface of D1-D2. Top: D2 (cyan) shown as B-factor defined coil with increased mobility indicated by thicker coil. D1 shown as green surface, with residues contacting D2 colored orange. Middle: Inverse display of (top). Inset: Sigma-A weighted electron density mesh (orange) contoured at 1.5 sigma for a loop of D2 (cyan) that protrudes into D1 (green).
Fig 3.
Comparative Tp0624 domain analyses.
(A) Left: overlay of Tp0624 D3 (purple) on the OmpA-like domain of Acinetobacter baumannii (grey, PDB ID 3TD5) in complex with a peptidoglycan derivative (light grey ball-and-stick colored by element). Right: Structure-based sequence alignment of Tp0624 with the peptidoglycan binding residues of A. baumanii OmpA-like domain. Critical residues are bolded and displayed in the inset panels. Inset: Zoomed-in view of the peptidoglycan binding site on A. baumanii OmpA-like domain (grey, left), with hydrogen bonds to the diaminopimelic acid residue shown as dotted lines. Star indicates the loop that comprises the outer side of the pocket. Contrast with the same view of Tp0624 D3, which despite conservation of the essential Arg, shows the loss of a critical Asp to Ala, the displaced position of the outer loop (star), and the large residues (Leu/His) in the lower loop that project into the binding site. (B) Electrostatic surface of Tp0624 D3 in same orientation as (A) shows a clear basic patch comprising the putative ligand binding site. (C) Overlay of Tp0624 D1 (green) on the E. coli trigger factor (grey, PDB ID 1P9Y). Grey bracket, region of the trigger factor that has been functionalized for ribosome binding and is not conserved in Tp0624 D1. Green bracket, region of Tp0624 D1 that has been extended to form the interfaces with D2 and D3. (D) Tp0624 D2 is a previously uncharacterized domain showing no significant structural similarity to any domain in the PDB. Tp0624 consists of a four-stranded parallel top sheet (cyan), four-stranded mixed bottom sheet (orange), and flanking helices (yellow). Left: orthogonal views of Tp0624 D2 tertiary structure. Right: Topology diagram of Tp0624 D2.
Fig 4.
Tp0624 amino acid sequence-based modularity analysis.
Orthologs of Tp0624 were identified using BLASTp. Tp0624 signal peptide (SP), domains (D1, green; D2, cyan; D3, purple), and linkers (L1 and L2, yellow) are shown. Indicated on the right are the numbers of orthologs with different domain combinations from spirochetes (left number) or other bacteria (right number). Sequences homologous to D1 and D2 only occur together, while D3 homologs are ubiquitous, and all three domains only occur together in treponemes (shown by an asterisk).
Fig 5.
Model of the potential roles of Tp0624 in T. pallidum peptidoglycan binding and cell envelope structure stabilization.
(Left) OmpA-like model: Tp0624 is exported across the inner membrane (IM) followed by cleavage and removal of the signal peptide. Tp0624 binds peptidoglycan (PG) via non-covalent interactions (orange arrow) involving the OmpA-like D3 while D1 and/or D2 interact with outer membrane-localized proteins (OMP). Residues from the treponemal conserved TAD(x)6E/QxxLSxxRA motif (putative non-canonical peptidoglycan binding motif) and surrounding pocket located within D3 are shown in orange (Right) MotB-like model: Tp0624 is translocated across the inner membrane, however the signal peptide is not cleaved and the protein remains anchored to the inner membrane, but with the functional head group directed into the periplasm. Tp0624 binds peptidoglycan via its OmpA-like domain, forming a structural bridge with the inner membrane. In this subcellular location, Tp0624 could function in cell envelope and/or flagellar stabilization.