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. 2007 Jan 30;104(5):1673-8.
doi: 10.1073/pnas.0609535104. Epub 2007 Jan 23.

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system

Richard Bayliss  1 , Richard Harris, Loic Coutte, Amy Monier, Remi Fronzes, Peter J Christie, Paul C Driscoll, Gabriel Waksman
Affiliations

NMR structure of a complex between the VirB9/VirB7 interaction domains of the pKM101 type IV secretion system

Richard Bayliss et al. Proc Natl Acad Sci U S A. .

Abstract

Type IV secretion (T4S) systems translocate DNA and protein effectors through the double membrane of Gram-negative bacteria. The paradigmatic T4S system in Agrobacterium tumefaciens is assembled from 11 VirB subunits and VirD4. Two subunits, VirB9 and VirB7, form an important stabilizing complex in the outer membrane. We describe here the NMR structure of a complex between the C-terminal domain of the VirB9 homolog TraO (TraO(CT)), bound to VirB7-like TraN from plasmid pKM101. TraO(CT) forms a beta-sandwich around which TraN winds. Structure-based mutations in VirB7 and VirB9 of A. tumefaciens show that the heterodimer interface is conserved. Opposite this interface, the TraO structure shows a protruding three-stranded beta-appendage, and here, we supply evidence that the corresponding region of VirB9 of A. tumefaciens inserts in the membrane and protrudes extracellularly. This complex structure elucidates the molecular basis for the interaction between two essential components of a T4S system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Overview of TraOCT/TraN complex structure. (A) Schematic topology diagram of TraOCT with β-strands color-ramped as in SI Fig. 7C. The 310 helix is colored white. TraN is represented as a yellow line crossing strands β1, β2, and β9 of TraOCT. Residues Trp-27 and Val-33, two major contact points with TraOCT, are shown. Strands β4 and β6 are shown at an angle to their adjacent strands to reflect their weaker H-bonding interactions. (B) Rotation of the model through 180° about the vertical axis with respect to C to show strands β4, β5, and β6 on the opposite face to the TraN-binding site. Residues N214 and 224 (N216 and N226 in A. tumefaciens) VirB9 are shown in ball-and-stick representation. (C) Stereo diagram of structure showing TraOCT in cartoon representation with loops, 310 helix, and β-strands colored as in A. TraN is shown as a stick model with carbon atoms colored yellow, nitrogen atoms colored blue, and oxygen atoms colored red. The same representations and color scheme is used in B and C. PyMOL was used for all structure figures (www.pymol.org).
Fig. 2.
Fig. 2.
Sequence conservation of the VirB9–VirB7 interaction site. (A) Sequence alignment of VirB9 homologs: pKM101 TraO (pKM101, NP#511196); A. tumefaciens VirB9 (A. tume, NP#396496); B. suis VirB9 (B. suis, NP#699268); pSB102 TraK (pSB102, NP#361041); Bordetella pertussis VirB9 (B. pert, NP#882293); Actinobacillus actinomycetemcomitans magB09 (A. acti, NP#067575); pR388 TrwF (pR388, CAA57030); Bartonella henselae TrwF (B. hens, CAF28337). Identical residues are shaded red, and conserved residues are shaded pink. The portion of the sequence modeled as structure is shown as a dashed line above the sequence, gray boxes containing colored arrows show the β-strands, and the 310 helix is shown as a black box. (B) Sequence alignment of VirB7 homologs: pKM101 TraN (pKM101, NP#511194); A. tumefaciens VirB7 (A. tume, NP#536291); B. suis VirB7 (B. suis, AAN33275); pSB102 TraI (pSB102, NP#361043); Bordetella pertussis VirB7 (B.pert, NP#882291); Actinobacillus actinomycetemcomitans magB07 (A. acti, NP#067577); pR388 TrwH (pR388, FAA00034); B. henselae TrwH (B. hens, AAM82208). VirB7 homologs were manually aligned based on the lipid-modified cysteine (green), the position of the A. tumefaciens cysteine, and the conserved “P[ILV]NK” VirB9-interaction motif (cyan). The portion of the sequence modeled as structure is shown as a dashed line above the alignment. In A and B, the cysteine residues involved in A. tumefaciens VirB9–VirB7 disulfide bond formation are colored orange, and key residues mutated in the A. tumefaciens proteins are shown in bold and underlined.
Fig. 3.
Fig. 3.
Structural detail of the TraOCT–TraN interaction site. (A) Detail of the interaction between TraN residues 23–27 and TraOCT. TraOCT main chain is depicted as a cartoon colored according to the scheme in Fig. 1. TraN and interacting side chains of TraOCT are shown as sticks colored white or yellow for carbon atoms in TraOCT or TraN, respectively, blue for nitrogen, red for oxygen, and orange for sulfur. (B) Detail of the interaction between TraN residues 31–34 and TraOCT using the same representation as A. (C) Surface representation of TraOCT colored white for no/low conservation, magenta for high conservation, and red for identical residues as defined by Fig. 2A. Identical residues on the surface of TraOCT and V33 of TraN are labeled. (D) Surface representation of modeled A. tumefaciens VirB9CT with the backbone ribbon of TraN superposed, shown in the same orientation as the pKM101 structure in C. VirB9CT is colored white for carbon, blue for nitrogen, red for oxygen, and orange for sulfur. Potential locations of VirB7 cysteine residue 24 are marked with orange spheres, and the “PLN” sequence predicted to bind to the hydrophobic pocket is marked as cyan backbone.
Fig. 4.
Fig. 4.
Conformational changes in VirB9 upon binding of TraN. TraOCT shows no global conformational change upon TraN binding. Differences in the average chemical shift 1H-15N HSQC (Δδavg) are plotted as a color ramp on the surface of TraOCT. Blue surface represents residues that exhibit Δδavg <0.1ppm, and red denotes Δδavg >0.9ppm or ablation of cross peak intensity. (A) Difference between 15N-labeled TraOCT monomer alone and bound to unlabeled TraN, shown from the same view as Fig. 1C. (B) Difference between 15N-labeled TraOCT monomer and dimer shown from same view as in A. TraN is shown to indicate the partial overlap of residues at the TraN interface with those exhibiting a high Δδavg.
Fig. 5.
Fig. 5.
Surface accessibility of VirB9 Cys substitution mutations and FLAG epitope tagged VirB9 derivatives. (A) Intact cells (Upper) and spheroplasts (Lower) were reacted with anti-FLAG antibodies and examined by immunofluorescence microscopy. Strains: B9, PC1009 with pBLC373 producing native VirB9; 216 & 226, PC1009 producing VirB9FL derivatives with FLAG epitope at these residues. (B) (Upper) Intact cells were incubated with the Cys reactive reagent MPB with (+) or without (-) preblocking with AMS. (Lower) Intact cells were untreated (-) or treated (+) with mPEG-maleimide (mPEG) (5 kDa). Strains: ΔB9, PC1009; ΔB9(B9), PC1009(pBLC373) producing native VirB9; PC1009 with pBLC373 derivatives carrying Cys substitutions at positions indicated.
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References

    1. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E. Annu Rev Microbiol. 2005;59:451–485. - PMC - PubMed
    1. Yeo HJ, Waksman G. J Bacteriol. 2004;186:1919–1926. - PMC - PubMed
    1. Schroder G, Lanka E. Plasmid. 2005;54:1–25. - PubMed
    1. Cascales E, Christie PJ. Nat Rev Microbiol. 2003;1:137–150. - PMC - PubMed
    1. Christie PJ. Biochem Biophys Acta. 2004;1694:219–234. - PMC - PubMed

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