Architecture of the Bacterial Flagellar Distal Rod and Hook of Salmonella

The bacterial flagellum is a large molecular complex composed of thousands of protein subunits for motility. The filamentous part of the flagellum, which is called the axial structure, consists of the filament, the hook, and the rods, with other minor components-the cap protein and the hook associated proteins. They share a common basic architecture of subunit arrangement, but each part shows quite distinct mechanical properties to achieve its specific function. The distal rod and the hook are helical assemblies of a single protein, FlgG and FlgE, respectively. They show a significant sequence similarity but have distinct mechanical characteristics. The rod is a rigid, straight cylinder, whereas the hook is a curved tube with high bending flexibility. Here, we report a structural model of the rod constructed by using the crystal structure of a core fragment of FlgG with a density map obtained previously by electron cryomicroscopy. Our structural model suggests that a segment called L-stretch plays a key role in achieving the distinct mechanical properties of the rod using a structurally similar component protein to that of the hook

Structural Analysis of the Essential Resuscitation Promoting Factor YeaZ Suggests a Mechanism of Nucleotide Regulation through Dimer Reorganization

Extent: 8p.Background: The yeaZ gene product forms part of the conserved network YjeE/YeaZ/YgjD essential for the survival of many Gram-negative eubacteria. Among other as yet unidentified roles, YeaZ functions as a resuscitation promoting factor required for survival and resuscitation of cells in a viable but non-culturable (VBNC) state. Methodology/Principal Findings: In order to investigate in detail the structure/function relationship of this family of proteins we have performed X-ray crystallographic studies of Vibrio parahaemolyticus YeaZ. The YeaZ structure showed that it has a classic actin-like nucleotide-binding fold. Comparisons of this crystal structure to that of available homologues from E. coli, T. maritima and S. typhimurium revealed two distinctly different modes of dimer formation. In one form, prevalent in the absence of nucleotide, the putative nucleotide-binding site is incomplete, lacking a binding pocket for a nucleotide base. In the second form, residues from the second subunit complete the nucleotide-binding site. This suggests that the two dimer architectures observed in the crystal structures correspond to a free and a nucleotide-bound form of YeaZ. A multiple sequence alignment of YeaZ proteins from different bacteria allowed us to identify a large conserved hydrophobic patch on the protein surface that becomes exposed upon nucleotide-driven dimer re-arrangement. We hypothesize that the transition between two dimer architectures represents the transition between the ‘on’ and ‘off’ states of YeaZ. The effect of this transition is to alternately expose and bury a docking site for the partner protein YgjD. Conclusions/Significance: This paper provides the first structural insight into the putative mechanism of nucleotide regulation of YeaZ through dimer reorganization. Our analysis suggests that nucleotide binding to YeaZ may act as a regulator or switch that changes YeaZ shape, allowing it to switch partners between YjeE and YgjD.Inci Aydin, Yumiko Saijo-Hamano, Keiichi Namba, Connor Thomas and Anna Roujeinikov

International journal of precision engineering and manufacturing : IJPEM

The CagA protein of Helicobacter pylori is associated with increased virulence and gastric cancer risk. CagA is translocated into the host cell by a H. pylori type IV secretion system via mechanisms that are poorly understood. Translocated CagA interacts with numerous host factors, altering a variety of host signalling pathways. The recently determined crystal structure of C-terminally-truncated CagA indicated the presence of two domains: the smaller, flexible N-terminal domain and the larger, middle domain. In this study, we have investigated the conformation, oligomeric state and stability of the N-terminal, middle and glutamate-proline-isoleucine-tyrosine-alanine (EPIYA)-repeats domains. All three domains are monomeric, suggesting that the multimerisation of CagA observed in infected cells is likely to be mediated not by CagA itself but by its interacting partners. The middle and the C-terminal domains, but not the N-terminal domain, are capable of refolding spontaneously upon heat denaturation, lending support to the hypothesis that unfolded CagA is threaded C-terminus first through the type IV secretion channel with its N-terminal domain, which likely requires interactions with other domains to refold, being threaded last. Our findings also revealed that the C-terminal EPIYA-repeats domain of CagA exists in an intrinsically disordered premolten globule state with regions in PPII conformation--a feature that is shared by many scaffold proteins that bind multiple protein components of signalling pathways. Taken together, these results provide a deeper understanding of the physicochemical properties of CagA that underpin its complex cellular and oncogenic functions

Crystallization and preliminary X-ray analysis of the C-terminal cytoplasmic domain of FlhA, a membrane-protein subunit of the bacterial flagellar type III protein-export apparatus

Crystals of the cytoplasmic domain of FlhA, a membrane-protein component of the bacterial flagellar type III protein-export apparatus from Salmonella, have been obtained and characterized by X-ray diffraction

Role of the C-Terminal Cytoplasmic Domain of FlhA in Bacterial Flagellar Type III Protein Export▿

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhAC) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhAC. The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhAC consists of at least three subdomains, a flexible linker, FlhACN, and FlhACC, and that FlhACN becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhAC not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure

A: Comparison of form 1 and form 2 dimers observed in the crystal structures of <i>Vp</i>YeaZ, <i>St</i>YeaZ, <i>Ec</i>YeaZ and <i>Tm</i>YeaZ.

<p>B: Location of residues L40, L47, I74, I78, L82 (shown in red) at the interface of <i>Vp</i>YeaZ dimer (left) and positions of the equivalent residues in <i>Tm</i>YeaZ form 2 dimer (right). The residue positions are shown for one half of the dimer, with the other half represented by its molecular surface. C: A stereo diagram showing modeled positions of a nucleotide molecule in the interdomain cleft of the form 1 (left) and the form 2 dimers. The nucleotide (ATP) molecule has been positioned based on structural similarity between domains I in YeaZ and Kae1. The modelled nucleotide is shown in ball representation. In the model of the form 2 dimer complex one of the ATP molecules is shown in light grey for clarity of illustration.</p

A sequence alignment of YeaZ homologues in <i>V. parahaemolyticus</i> (V. par), <i>S. typhimurium</i> (S. typ), <i>E. coli</i> (E. col), <i>T. maritima</i> (T. mar), <i>Pasteurella multocida</i> (P. mul), <i>Xylella fastidiosa</i> (X. fas), <i>Sinorhizobi

<p>The elements of the secondary structure and the sequence numbering for <i>Vp</i>YeaZ are shown above the alignment. The fully conserved residues are shown by a black box with reverse type. The position of the conserved and semi-conserved residues which can be identified in the nucleotide-binding site of proteins belonging to the ASKHA family, are highlighted by black and gray boxes respectively, and marked with “n” underneath the aligned sequences. The residues forming a hydrophobic surface patch which becomes exposed upon dimer re-organisation are enclosed by a box and marked with “<i>h</i>” underneath the alignment. Alignment was carried out using the ClustalW server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023245#pone.0023245-Thompson1" target="_blank">[16]</a>. The figure was created using ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023245#pone.0023245-Gouet1" target="_blank">[17]</a>.</p

The structural overlap between <i>Vp</i>YeaZ (green), <i>Tm</i>YeaZ (purple), <i>Ec</i>YeaZ (red) and <i>St</i>YeaZ (black).

<p>The side chains of the conserved and semi-conserved residues likely to be implicated in nucleotide binding are shown for <i>Vp</i>YeaZ.</p

A: Stereo diagram of the structure of the <i>Vp</i>YeaZ monomer.

<p>Each element of the secondary structure is labeled. Domains I and II and the putative nucleotide-binding cleft are identified. The figure was prepared using PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023245#pone.0023245-Delano1" target="_blank">[11]</a>. B: The topology of the secondary structure elements. Residue numbers are indicated at the start and end of each secondary structure element. C: SDS-PAGE showing the time-course of VpYeaZ digestion by Glu-C protease. The positions of molecular mass markers are shown to the left. The arrow indicates a relatively stable C-terminally truncated fragment. D: Amino acid sequence of VpYeaZ with Glu-C-sensitive site identified in this study shown by the arrow.</p