On the quaternary association of the type III secretion system HrcQB-C protein: experimental evidence differentiates among the various oligomerization models.
The HrcQB protein from the plant pathogen Pseudomonas syringae is a core component of the bacterial type III secretion apparatus. The core consists of nine proteins widely conserved among animal and plant pathogens which also share sequence and structural similarities with proteins from the bacterial flagellum. Previous studies of the carboxy-terminal domain of HrcQB (HrcQB-C) and its flagellar homologue, FliN-C, have revealed extensive sequence and structural homologies, similar subcellular localization, and participation in analogous protein-protein interaction networks. It is not clear however whether the similarities between the two proteins extend to the level of quaternary association which is essential for the formation of higher-order structures within the TTSS. Even though the crystal structure of the FliN is a dimer, more detailed studies support a tetrameric donut-like association. However, both models, dimer and donut-like tetramer, are quite different from the crystallographic elongated dimer of dimers of the HrcQB-C. To resolve this discrepancy we performed a multidisciplinary investigation of the quaternary association of the HrcQB-C, including mass-spectrometry, electrophoresis in non-reductive conditions, gel filtration, glutaraldehyde cross-linking and small angle X-ray scattering. Our experiments indicate that stable tetramers of elongated shape are assembled in solution, in agreement with the results of crystallographic studies. Circular dichroism data are consistent with a dimer-dimer interface analogous to the one established in the crystal structure. Finally, molecular dynamics simulations reveal the relative orientation of the dimers forming the tetramers and the possible differences from that of the crystal structure.
Department of Biology, University of Crete, P.O. Box 2208, GR-71409 Heraklion, Crete, Greece.
Vasiliki E
VE
Fadouloglou
Marina N
MN
Bastaki
Alison E
AE
Ashcroft
Simon E V
SE
Phillips
Nicholas J
NJ
Panopoulos
Nicholas M
NM
Glykos
Michael
M
Kokkinidis
D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003198:Computer Simulation; D008958:Models, Molecular; D055503:Protein Multimerization; D053838:Scattering, Small Angle; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; C441634:HrcQb protein, Pseudomonas syringae