Table 1.
Assignment and structural statistics for the solution structure of VirB7XAC2622_24–139.
Figure 1.
NMR and X-ray models of VirB7XAC2622.
Superposition of 20 lowest energy VirB7XAC2622_24–139 solution structures: (A) backbone traces of the full-length protein (residues 24–139) and (B) ribbon representation of the folded domain (residues 51–134) in the NMR ensemble. (C) Ribbon representation of the X-ray crystal structure of VirB7XAC2622_51–134. Water molecules and one isopropanol ligand are depicted. (D) Superposition of the X-ray (green) and NMR (lowest energy model; red) structures of the VirB7XAC2622 globular domain.
Table 2.
Crystallographic data collection and refinement statistics of VirB7XAC2622_51–134.
Figure 2.
VirB7XAC2622_24–139 oligomerization.
(A) Superposition of 15N-HSQC spectra of 15N-VirB7XAC2622_24–139 at different concentrations. Cyan: 850 µM; blue: 600 µM; green: 400 µM; yellow: 300 µM; orange: 200 µM; pink: 100 µM; red: 50 µM; purple: 25 µM; brown: 13 µM and black: 7 µM. The inserts are amplifications of selected spectral regions showing peak movements as the protein concentration varies from 850 to 7 µM in the direction of the arrows. (B) Weighted chemical shift changes (Δδcomp) observed upon dilution from 100 µM to 7 µM. (C) Surface representation of VirB7XAC2622_24–139 colored according to the weighted chemical shift changes. Residues involved in oligomerization are located in the folded domain and in the unfolded N-terminus. 1H-15N correlation of Leucine 89 (L89) was not detected in any 15N-HSQC spectrum probably due to chemical exchange. (D) Ribbon representations of docking models of the VirB7XAC2622 – VirB7XAC2622 interaction. Helices, β-strands and coil regions are colored red, yellow and green, respectively. Residues involved in intermolecular NOEs are shown as stick models colored brown (residues A43, T45, E46, I47, L49) and green (T63, S85, D86, Y87, T88, I90). Six models are shown.
Figure 3.
The VirB7XAC2622 N-terminus recognizes the VirB9XAC2620 C-terminal domain.
(A) Superposition of the 15N-HSQC spectra of 15N-VirB7XAC2622_24–139 alone (orange) and in the presence of 14N-VirB9XAC2620_154–255 (green). (B) Same as A, but with a close-up view of the central spectral region. The residues for which significant changes in peak positions were observed upon complex formation are indicated. (C) Weighted chemical shift changes (Δδcomp) of VirB7XAC2622 upon binding to VirB9XAC2620. (D) Residues affected by interaction with VirB9XAC2620_154–255 (residues 27–41; red) are color coded on the structure of VirB7XAC2622_24–139.
Figure 4.
The conformation of the VirB9XAC2620 C-terminal domain changes significantly upon interacting with VirB7XAC2622.
15N-HSQC spectra of 15N-VirB9XAC2620_154–255 in the absence (red) and in the presence (green) of 14N-VirB7XAC2622_24–139. Spectra were collected at 30°C on a 500 MHz spectrometer equipped with a room temperature probe.
Figure 5.
15N relaxation data for VirB7XAC2622_24–139.
(A) Heteronuclear {1H}-15N NOE (HetNOE), (B) 15N-T1 and (C) 15N-T2 relaxation rates as a function of the protein sequence. Black squares: 15N-VirB7XAC2622_24–139 at 800 µM; red circles: 15N-VirB7XAC2622_24–139 at 100 µM; blue inverted triangles: 15N-VirB7XAC2622_24–139 – 14N-VirB9XAC2620_154–255 complex at 400 µM. Data are presented as mean ± uncertainty of the fitted parameter. Relaxation parameters for residues involved in oligomerization (43–47 and some between positions 86 and 93) were not obtained because they were not detectable in the 15N HSQC spectrum at high protein concentration (800 µM) and display amplitudes too low to allow precise measurements at lower concentrations.
Figure 6.
A complex between VirB7XAC2622, VirB9XAC2620 and VirB10XAC2619 is formed in vivo in Xac cells.
(A) Immunoblot analysis of VirB9XAC2620, VirB7XAC2622 and VirB10XAC2619 protein levels in bacterial total protein extracts. Polyclonal antibodies raised against VirB9XAC2620 (α-VirB9XAC2620), VirB7XAC2622 (α-VirB7XAC2622) and VirB10XAC2619 (α-VirB10XAC2619) were used. Rec: purified recombinant proteins used as positive controls; WT: Wild type Xac strain; ΔvirB7: ΔvirB7XAC2622 knockout strain and ΔvirB7+pVirB7: ΔvirB7XAC2622 knockout cells complemented with the pUFR-VirB7 plasmid. Recombinant VirB7XAC2622_24–139 has a slightly greater electrophoretic mobility than native VirB7XAC2622, possibly due to the presence of a covalently attached lipid moiety or retention of the signal sequence of the native protein. VirB9XAC2620_34–255 (26 kDa) and VirB10XAC2619_85–389_His (34 kDa) are also smaller than the respective native proteins. (B) Coimmunoprecipitation assays detecting reciprocal interactions between VirB proteins. Xac total cell lysates were immunoprecipitated (IP) with anti-VirB7XAC2622, anti-VirB9XAC2620 or anti-VirB10XAC2619 serum and the presence of VirB7, VirB9 or VirB10 was detected by immunoblot analysis. Lane labels are the same as in part (A). No VirB proteins were detected in control experiments using pre-immune sera (data not shown). (C) Characterization of the ΔvirB7XAC2622 gene knockout in Citrus sinensis infections. Macroscopic symptoms on abaxial surface of orange leaves 12 days post inoculation with wild type (left) and ΔvirB7 (right) strains of Xac. (D) Growth curves of Xac strains on orange leaves. Blue: wild type; red: ΔvirB7. Data are presented as mean ± standard deviation.
Figure 7.
VirB7XAC2622 structure and interactions in the context of the T4SS outer membrane complex.
(A) Schematic organization of the VirB7XAC2622 protein, based on bioinformatics and experimental data. (B) Sequence alignment of the VirB7 proteins from Xanthomonas citri subsp. citri (GenBank code AAM37471), Neisseria flavescens (EER57212), Neisseria sp. oral taxon 014 str. F0314 (EFI23373) and TraN from pKM101 (AAA86456). Sequences begin at the cysteine residues of the predicted lipidation sites. Black asterisks indicate residues involved in observed intermolecular NOEs. Blue asterisks indicate the PVNK motif found in the pKM101 TraN protein. Blue brackets above and below the alignment indicate the VirB9XAC2620 and TraO binding sites in VirB7XAC2622 and TraN, respectively. The black bracket indicates the N-terminal oligomerization site. Residues outlined in red are conserved in at least three of the four sequences. Note that the codon for V55 of the protein from Neisseria flavescens is incorrectly assigned as a start codon in the GenBank database. (C) Final molecular dynamics configuration of the (VirB7XAC2622_37–139-TraOCT)14 complex. TraOCT is represented in red, VirB7XAC2622 is in cyan (residues 37–41), magenta (residues 42–49) and dark blue (residues 50–139). One VirB7XAC2622_37–139 unit is shown in green. (D) Putative model for the VirB7XAC2622-VirB9XAC2620-VirB10XAC2619 outer membrane complex produced by adding TraN residues 19–32 (representing VirB7XAC2622 residues 22–36, see alignment in part B) and TraFCT (representing VirB10XAC2619) to the structure shown in part C. The color scheme is the same as in part C except that cyan represents VirB7XAC2622 residues 22–41 and TraF/VirB10 is shown in yellow. One complete VirB7XAC2622 molecule is shown in green.
Figure 8.
Superposition of the structure of the VirB7XAC2622 N0 domain with structural homologs.
The VirB7XAC2622 globular domain (residues 51–134; green) is superposed with (A) the signaling domain of PupA (orange, residues 3–76, PDB: 2A02); (B) the N0 domain of GspD (red, amino acids 3–79, PDB: 3EZJ); (C) the N0 domain of EscC (yellow, amino acids 31–103, PDB: 3GR5), (D) residues 97–176 of gene product 44 from bacteriophage Mu (light blue, PDB: 1WRU) and (E) residues 81–161 of DotD from L. pneumophila (purple, PDB: 3ADY).
Table 3.
Structural similarity of the VirB7XAC2622 globular domaina.