Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICE St3 From Streptococcus thermophilus

Abstract

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

Keywords: Gram - positive bacteria; Integrative and Conjugative Element (ICE); VirB8-like proteins; conjugation; type IV secretion system.

FIGURE 1

OrfG belongs to the TcpC superfamily (A) Schematic representation of OrfG, TraM and TraH subdomains organization. Boundaries of OrfG subdomains and TraM and TraH NTF2-like domains are stated. TMD for transmembrane domain; CC for coiled-coil domain (B) Phylogenetic tree of representative members from the TcpC superfamily including OrfG. This tree was inferred by using the Maximum Likelihood method based on the JTT matrix-based model (Guindon et al., 2010). The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured as the number of substitutions per site. All positions containing gaps and missing data were eliminated. There was a total of 284 positions in the final dataset. Evolutionary analyses were conducted using MEGA7 (Tamura and Nei, 1993).

FIGURE 2

Structures of OrfG_64–204, TcpC_104–231, TraM_190–322, and TraH_57–183. From left to right respectively, the four monomers (PDB id 6zgn, 4ec6, 3ub1, and 5aiw) are displayed in the same orientation and represented as rainbow-colored cartoons, with the N-terminal end of the polypeptide chain in blue and the C-terminal end in red (A). Their topology is schematized with the same rainbow-color code and the name of the secondary structures (B).

FIGURE 3

Comparison of 17 representative three-dimensional structures of VirB8 and VirB8-like proteins. The structure of OrfG_64–204 (PDB id 6zgn) is represented as a cartoon (A), with its amino acids colored according to the Consurf server (Ashkenazy et al., 2016) which analyzed the similarity calculated in the sequence alignment deduced from the structure superposition produced by mTM-Align (Dong et al., 2018a) for a set of sventeen selected proteins. The amino acid shown with sticks (Val191 in OrfG) and localised in β₄ facing α ₁ is the only one whose side chain shares a similar nature in all structures, while there is no conserved residue. The 17 structures (PDB ids 2cc3, 3ub1 (limited to TcpC_104–231), 3wz3, 3wz4, 4akz, 4ec6, 4jf8, 4kz1, 4lso, 4mei, 4nhf, 4o3v, 5aiw, 5cnl, 5i97, 6iqt, 6zgn) are represented (B) as superposed wires (except OrfG as a cartoon) using a color-code based on the quality of the superposition of these structures. For each position, a gradient from violet to red is used depending on the low to high values of the root mean square calculated on the distance between all the possible pairs of the 17 superposed Cα carbons at this position (grey means that mTM-Align found at least one structure impossible to align). This representation highlights the resemblances and differences in the NTF2-like folds of these proteins.

FIGURE 4

Structural and phylogenetic trees of Gram-negative VirB8 and Gram-positive VirB8-like proteins. (A) Structure-based tree derived from the optimized superposition of the atomic coordinates (left). 17 representative structures were retrieved from the PDB and compared by mTM-align (Dong et al., 2018a). The name of the protein, its source and PDB id are indicated for each protein. Members of class I⁻ are in green, class II⁻ in red, class III⁻ in blue, and class I⁺ in orange where the (⁻) and (⁺) correspond to VirB8-like structures from Gram-negative and -positive bacteria, respectively. (B) Secondary structure topology observed within each class is schematized at the bottom of the figure. Among the proteins compared in the structural analysis, all the sequences that belong to the PFAM group PF04335 were subjected to multiple sequence alignment and neighbor-joining tree building. The result is shown on the right-hand side of the figure. Numbers at nodes indicate the bootstrap values as percentage (1,000 replicates). Scale bar indicates the number of amino acid differences per site.

FIGURE 5

Assemblies of Gram-positive VirB8-like proteins observed in the crystals. A crystallographic 2-fold axis relates two monomers of OrfG_64–204 (PDB id 6zgn) (A), while TraM_190–322 (4ec6) (B) and TcpC_99–359 (3ub1) (C) form trimers. Each assembly is shown perpendicular (top) and parallel (bottom) to its symmetry axis. The interface with the highest surface area, here represented for OrfG_64–204, is resolutely rejected by PISA (Krissinel and Henrick, 2007). The interface (*1) in TraM_190–322 extends on ∼300 Å² and involves two to nine hydrogen bonds or salt bridges depending on the observed monomer (as calculated by PISA for residues 214 to 318 of 4ec6). The equivalent interface in TcpC_99–359 (*2) represents ∼430 Å² (PISA on residues 104–228 of 3ub1) and six to nine hydrogen bonds or salt bridge depending on the monomer. It is by far slighter than the interface (*3) observed between the central domain (TcpC_104–231) of one monomer and the C-terminal domain (TcpC_239–359) of a second one, which extends on ∼980 Å² and involves 13–14 hydrogen bonds or salt bridges depending on the observed monomer. The structure-based sequence alignment of OrfG, TcpC and TraM available in Figure 6 shows residues involved in these interactions.

FIGURE 6

Structure-based sequence alignment of three Gram-positive VirB8-like proteins. The sequence alignment was generated by mTM-align and manually modified, based on the three structures of OrfG_64–204 (pdb entry 6zgn), TcpC_99–359 (3ub1) and TraM_190–322 (4ec6). Residues of OrfG_64–204 that were not observed in the electron density are shown as lowercases, as well as residues 205–327 equivalent to the C-terminal domain of TcpC and for which no structure is known yet. Numbering above the sequences corresponds to OrfG. Secondary structures are represented by arrows (β-strands) and squiggles (α-helices), in black when shared by OrfG and TcpC, in grey when only present in OrfG, and in cyan when only present in TcpC. Residues of TraM and TcpC involved in the trimer assemblies are highlighted, in orange in one partner and in blue in the facing monomer: white letters with dark background are for residues involved in hydrogen bonds in all interface instances in the asymmetric units, while dark letters with lighter background are involved in contact in at least one interface (medium background when at least 50% of the residue is buried in the contact, light background if less than 50% is buried). Residues of OrfG_64–204 that delineate the pocket in which an unknown ligand is bound (see Supplementary Figure S1) are marked with an overbar.

FIGURE 7

Analysis of oligomerization of native and truncated versions of OrfG_64–331 by in vitro chemical cross-linking and size exclusion chromatography. In vitro chemical cross-linking of OrfG_64–331 (1A), OrfG_64–204 (2A) and OrfG_223–331 (3A). SDS-PAGE analysis of 3 µM of the purified OrfG_64–331 and its truncated versions in absence or in presence of an increasing concentration (0.05–5%) of paraformaldehyde (PFA). Samples were loaded without heating treatment. The concentration of PFA used for each reaction is mentioned at the top of each column. Asterisks indicate the identified oligomers stabilized by PFA cross-linking. Size exclusion chromatography (SEC) of the purified OrfG_64–331 (1B), OrfG_64–204 (2B) and OrfG_223–331 (3B) using Superdex 200 16/600. The elution volume (ml) is plotted on the x-axis and the 280-nm absorbance is plotted on the y-axis. SDS-PAGE analysis of SEC purification of OrfG_64–331 (1C), OrfG_64–204 (2C) and OrfG_223–331 (3C). Red and green lines correspond to the SEC fractions selected from each peak and analyzed by SDS-PAGE. Electrophoretic separation shows that all identified peaks contain exclusively the analyzed protein. Molecular weight markers (in kDa) are indicated on the left of each SDS-PAGE pattern. Protein bands corresponding to each protein are indicated by black arrows.

FIGURE 8

SEC-MALS analysis. Elution profile (black lines) of OrfG_64–331 (A), OrfG_64–204 (B), OrfG_223–331 (C) and OrfG_64–215 (D) are shown with the molecular weight calculated by MALS (gray lines). The elution time (min) is plotted on the x-axis. The molar mass (in logarithmic scale) is plotted in the first y-axis and the refractive index is plotted in the second y-axis. The molecular weight (in kDa) and the contribution in mass fraction (in %) of each visible peak are shown at the top of the corresponding peak.