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. 2004 Jan 6;101(1):70-5.
doi: 10.1073/pnas.0304579101. Epub 2003 Dec 23.

Structure of HrcQB-C, a conserved component of the bacterial type III secretion systems

Vasiliki E Fadouloglou  1 , Anastasia P Tampakaki, Nicholas M Glykos, Marina N Bastaki, Jonathan M Hadden, Simon E Phillips, Nicholas J Panopoulos, Michael Kokkinidis
Affiliations

Structure of HrcQB-C, a conserved component of the bacterial type III secretion systems

Vasiliki E Fadouloglou et al. Proc Natl Acad Sci U S A. .

Abstract

Type III secretion systems enable plant and animal bacterial pathogens to deliver virulence proteins into the cytosol of eukaryotic host cells, causing a broad spectrum of diseases including bacteremia, septicemia, typhoid fever, and bubonic plague in mammals, and localized lesions, systemic wilting, and blights in plants. In addition, type III secretion systems are also required for biogenesis of the bacterial flagellum. The HrcQ(B) protein, a component of the secretion apparatus of Pseudomonas syringae with homologues in all type III systems, has a variable N-terminal and a conserved C-terminal domain (HrcQ(B)-C). Here, we report the crystal structure of HrcQ(B)-C and show that this domain retains the ability of the full-length protein to interact with other type III components. A 3D analysis of sequence conservation patterns reveals two clusters of residues potentially involved in protein-protein interactions. Based on the analogies between HrcQ(B) and its flagellum homologues, we propose that HrcQ(B)-C participates in the formation of a C-ring-like assembly.

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Figures

Fig. 3.
Fig. 3.
Sequence alignments. The numbering scheme corresponds to the sequence of the full-length HrcQB, and the secondary structural elements shown at Upper are those of the HrcQB-C structure. Alignment was performed with clustal w (18) and plotted with the espript program (19). Strictly conserved residues are highlighted, and similar residues are boxed. (A) Sequence alignment of the HrcQB-C and the C termini of homologous proteins identified by a blasta search with an identity of 26% or higher. Three pairs of sequences from plant pathogens, animal pathogens, and flagella are included. The pairs are chosen to be as different from each other as possible. The proteins aligned are listed in Methods. (B) Sequence alignment between the HrcQB-C and the C-terminal region (residues 154–235) of the HrcQA. Triangles indicate positions corresponding to HrcQB-C residues that participate in the dimer–dimer interface.
Fig. 1.
Fig. 1.
A schematic representation of the HrcQB-C structure. Each chain is individually colored and labeled. (A) A view of the complete tetramer. The dimers are related by a two-fold axis that is indicated by a red line. (B) A view of the tetramer along the two-fold axis. The molecule is rotated 90° relative to A. (C) A stereoview of the HrcQB-C dimer. The red line indicates the local two-fold axis. Symmetry related cysteines that form the disulfide bond are shown as a ball-and-stick model.
Fig. 2.
Fig. 2.
A stereoview of the 2Fo – Fc electron density map contoured at 1.5 σ with the final model overlaid on it. The region shown corresponds to part of the sheet formed by the strands β1 (chain A), β1 (chain B), and β3 (chain B).
Fig. 4.
Fig. 4.
Molecular surface of the HrcQB-C that is colored according to the degree of amino acid conservation as calculated by the 3D cluster analysis program 3dca (20). A multiple sequence alignment (identity of 47% or better) among the proteins P. syringae pv. syringae HrpU, P. syringae pv. tomato HrcQb, Pseudomonas fluorescens RscQB, E. amylovora HrcQ, and Pantoea agglomerans HrcQB was performed. The coloring code ranges from blue for nonconserved to orange for highly conserved residues. Three views of the molecule, related by rotations about a horizontal axis (indicated as a black line in A) are presented. (A) Side view. (B) Concave surface (rotated 90° clockwise relative to A). (C) Convex surface (rotated 90° counterclockwise relative to A).
Fig. 5.
Fig. 5.
HrcQB–HrcQA protein interactions. (A) Yeast two-hybrid analysis of HrcQB--HrcQA protein interaction (for details see Methods). Yeast cells expressing the hybrid proteins DBD-HrcQA and activation domain HrcQB (area 3) were grown in the absence of Ade and His and formed red colonies, as did the positive control cells, indicating that the hybrid proteins interact with each other. Plasmids pVA3-1 and pTD1-1 were used as positive controls, (area 2) and the vectors pAS2-1 and pACT2 without inserts served as negative controls (area 1). (B) HrcQB-C is involved in the association with the HrcQA. Wild-type and mutant HrcQA proteins were mixed in equimolar amounts with HrcQB-C and were incubated for 6 h at 4°C, then antibody specific for HrcQB (1:5,000) was added and was further incubated overnight at 4°C. Immunocomplexes were precipitated with protein A-agarose at 4°C overnight. The immunoprecipitates were separated by SDS/PAGE and visualized by immunoblotting with HrcQA-specific antibody. Lane 1, HrcQB; lane 2, HrcQA; lane 3, HrcQA-N (residues 1–65); lane 4, HrcQA-C (residues 66–238). Prestained molecular mass markers (M) in kDa are indicated on the left. The arrows indicate the HrcQA and HrcQA-C proteins that coimmunoprecipitated with HrcQB-C. (C) An autoradiogram that shows the binding of HrcQB to His-10-HrcQA protein and to the C-terminal truncated version. Lane 1, HrcQA; lane 2, HrcQA-N; lane 3, HrcQA-C. Far Western blot analysis was performed as described in Methods. The membrane was probed with 35S-labeled HrcQB and subjected to autoradiography. (D) The presence of proteins on the membrane shown in C was confirmed by Western blot analysis using anti-His-AP conjugate (1:1,000). Prestained molecular mass markers (M) in kDa are indicated on the left.
Fig. 6.
Fig. 6.
Subcellular localization of HrcQB and HrcQA proteins in P. syringae pv. Phaseolicola. (A) Cell-bound (C) and cell-free supernatant (S) fractions (see Methods) were subjected to SDS/PAGE and immunoblot analysis with antisera specific for HrcQA, HrcQB, HrpZ, and AvrPphB. The proteins HrpZ and AvrPphB are used as positive and negative controls (secreted and nonsecreted in culture), respectively. (B) The effects of washing with NaCl, urea, or Triton X-114 on the membrane association of HrcQA and HrcQB. P and S indicate the supernatants pellets and from ultracentrifugation, respectively (see Methods). Molar concentrations of NaCl, urea, and Triton X-114 are indicated above the respective panels.
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