The 2.5 Å structure of the enterococcus conjugation protein TraM resembles VirB8 type IV secretion proteins

Abstract

Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G-) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G-) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G- bacteria, we propose a new classification scheme of VirB8-like proteins.

FIGURE 1.

TraM localization and characterization. A, TraM localizes to the cell envelope of pIP501 harboring E. faecalis JH2-2 cells. The localization of TraM in the cell fractions was detected by Western blot with anti-TraMΔ antibodies. CW, cell wall; M, membrane; CP, cytoplasm. B, cross-linking assay of TraMΔ. Lane M, molecular mass standard; lane 1, control (no glutardialdehyde, no treatment); lanes 2–6, TraMΔ with 0/0.001/0.01/0.05/0.1% glutardialdehyde. C, opsonophagocytic killing and inhibition of killing assays using anti-TraMΔ antisera.

FIGURE 2.

The structure of TraM_214–322. A, cartoon representation of TraM_214–322 with view onto the twisted β-sheet and 90° turned about the 3-fold axis. Secondary structure elements are highlighted (helices in cyan and strands in purple). B, detailed view of the twist in strand 1. C, trimerization interface between β-strands 1 and 2 of chain F and α-helix 1 of chain D. D, one TraM trimer shown in cartoon representation. The monomers are colored green (chain D), red (chain E), and cyan (chain F), respectively. The monomer-monomer contact region is indicated. IS, interaction site. E, surface representation of the interaction area between monomers D and F. F, the C-terminal end of monomer F (stick representation) nestled in a hydrophobic cleft of monomer D (surface representation).

FIGURE 3.

The potential TraM coiled-coil motif. A, RMSD of the resulting backbone after least square fit to the starting model backbone. The results from GROMACS simulations (black lines) and Amber simulations (gray lines) are shown. The graphs are represented as progressive means of 25 data points. B, hydrophobic fraction of the solvent-accessible surface area per residue. The area remains stable in Amber03 (left panel) and GROMACS (right panel) simulations (three each). The data graphs are represented as progressive means of 50 data points. C, alignment of the three final Amber03 coiled-coil models in cartoon representation; the view is along the coiled-coil axis and 90° turned. D, highlighting on the hydrophobic residues (stick representation) facing toward the center of the triple coiled-coil model.

FIGURE 4.

Structural comparison of TraM_214–322 to related proteins. A, cartoon representation of TraMΔ, NTF2 (R. norvegicus, Protein Data Bank code 1OUN), the C-terminal and the central domain of TcpC (C. perfringens, Protein Data Bank code 3UB1), and the periplasmic domain of VirB8 from A. tumefaciens (Protein Data Bank code 2CC3) and B. suis (Protein Data Bank code 2BHM), respectively; secondary structure elements are highlighted (helices in cyan and strands in purple). B, comparison of the TraMΔ trimer to the TcpC trimer, formed by the central domains of TcpC monomers.

FIGURE 5.

Comparison of the domain arrangement of TraM and its structurally related proteins from G+ and G− putative T4SS. The amino acid sequence contained in the available structures is indicated by a dotted line below the individual representations. Because the structure of Orf13 from Tn916 is not available, the two potential domains are assigned according to secondary structure predictions with PsiPred. Transmembrane helices have been predicted for all proteins as described above. The potential coiled-coiled motifs of TraM and TcpC are highlighted as gray boxes (CC).