The type IV secretion system of Helicobacter pylori consists of 10--15 proteins responsible for transport of the transforming protein CagA into target epithelial cells. Secretion of CagA crucially depends on the hexameric ATPase, HP0525, a member of the VirB11-PulE family. We present the crystal structure of a binary complex of HP0525 bound to ADP. Each monomer consists of two domains formed by the N- and C-terminal halves of the sequence. ADP is bound at the interface between the two domains. In the hexamer, the N- and C-terminal domains form two rings, which together form a chamber open on one side and closed on the other. A model is proposed in which HP0525 functions as an inner membrane pore, the closure and opening of which is regulated by ATP binding and ADP release.
The coupling of ATP binding/hydrolysis to macromolecular secretion systems is crucial to the pathogenicity of Gram‐negative bacteria. We reported previously the structure of the ADP‐bound form of the hexameric traffic VirB11 ATPase of the Helicobacter pylori type IV secretion system (named HP0525), and proposed that it functions as a gating molecule at the inner membrane, cycling through closed and open forms regulated by ATP binding/hydrolysis. Here, we combine crystal structures with analytical ultracentrifugation experiments to show that VirB11 ATPases indeed function as dynamic hexameric assemblies. In the absence of nucleotide, the N‐terminal domains exhibit a collection of rigid‐body conformations. Nucleotide binding ‘locks’ the hexamer into a symmetric and compact structure. We propose that VirB11s use the mechanical leverage generated by such nucleotide‐dependent conformational changes to facilitate the export of substrates or the assembly of the type IV secretion apparatus. Bio chemical characterization of mutant forms of HP0525 coupled with electron microscopy and in vivo assays support such hypothesis, and establish the relevance of VirB11s ATPases as drug targets against pathogenic bacteria.
TraG-like proteins are potential NTP hydrolases (NTPases) that are essential for DNA transfer in bacterial conjugation. They are thought to mediate interactions between the DNA-processing (Dtr) and the mating pair formation (Mpf) systems. TraG-like proteins also function as essential components of type IV secretion systems of several bacterial pathogens such as Helicobacter pylori. Here we present the biochemical characterization of three members of the family of TraG-like proteins, TraG (RP4), TraD (F), and HP0524 (H. pylori). These proteins were found to have a pronounced tendency to form oligomers and were shown to bind DNA without sequence specificity. Standard NTPase assays indicated that these TraG-like proteins do not possess postulated NTP-hydrolyzing activity. Surface plasmon resonance was used to demonstrate an interaction between TraG and relaxase TraI of RP4. Topology analysis of TraG revealed that TraG is a transmembrane protein with cytosolic N and C termini and a short periplasmic domain close to the N terminus. We predict that multimeric inner membrane protein TraG forms a pore. A model suggesting that the relaxosome binds to the TraG pore via TraG-DNA and TraG-TraI interactions is presented.Bacterial conjugation is responsible for the spread of genetic traits among a broad range of bacterial species. It is the primary mechanism for dissemination of antibiotic resistance among human pathogens (60). Nearly all functions required to mediate bacterial conjugation are encoded by conjugative plasmids, which are usually further endowed with antibiotic resistance genes (64). In general, transfer (Tra) proteins are grouped into functional classes, defined as those involved in mating pair formation (Mpf) and DNA processing (Dtr). Secretion systems used by some pathogens, such as Agrobacterium tumefaciens, Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila, for delivering effector molecules to eukaryotic cells are composed of protein components evolutionarily related to those of Mpf complexes (12). Such macromolecular transfer systems ancestrally related to the conjugative Mpf complexes are called type IV secretion systems, as originally proposed by Salmond (46). Each of these systems secretes distinct DNA and/or protein substrates. TraG-like proteins (named for the protein of IncP plasmid RP4 [31]) are present in all conjugative DNA transfer systems and in several type IV secretion systems. Although TraG-like proteins are essential components in these secretion systems (12), their function remains poorly understood.The Dtr systems of conjugative plasmids are best characterized at the initiation stage of DNA processing. The relaxosome (20) is a complex of several Dtr proteins (relaxosomal proteins) bound to a specific DNA sequence, the origin of transfer (oriT) of the conjugative plasmid. This complex initiates DNA transfer by producing a single-stranded scission at the nic cleavage site within oriT. In this reaction, the catalytic key component, called the relaxase, becomes transiently ...
Type IV secretion systems mediate intercellular transfer of macromolecules via a mechanism ancestrally related to that of bacterial conjugation machineries. TraC of the IncN plasmid pKM101 belongs to the VirB5 family of proteins, an essential component of most type IV secretion systems. Here, we present the structure of TraC. VirB5͞TraC is a single domain protein, which consists of a three helix bundle and a loose globular appendage. Structure-based site-directed mutagenesis followed by functional studies indicates that VirB5 proteins participate in protein-protein interactions important for pilus assembly and function.conjugation ͉ crystal structure ͉ pilus ͉ TraC B acterial conjugation systems are macromolecular DNA transfer systems. Their impact on human health is highly significant as conjugation is the major vector for the dissemination of antibiotic resistance genes (1). Recent work has shown that several plant and human pathogens have evolved secretion machineries ancestrally related to conjugation systems for the purpose of delivering virulence effectors (proteins or protein-DNA complexes) to eukaryotic cell targets. Such pathogens include extracellular organisms such as Agrobacterium tumefaciens, the causative agent of crown gall disease, Bordetella pertussis, the agent responsible for whooping cough in children, and Helicobacter pylori, responsible for gastric ulcers and stomach cancer (reviewed in refs. 2-4). More recently, intracellular bacterial pathogens, such as Brucella suis, the causative agent for brucellosis, and Legionella pneumophila, the causative agent of legionnaire's disease, were shown to require such systems for their virulence (5, 6). Bacterial conjugation systems and their related counterparts in pathogenic bacteria are collectively known as type IV secretion systems (T4SS).T4SSs form supramolecular assemblies generally composed of a pilus assembly machinery͞secretion apparatus that presumably produces a channel for translocation of the effectors. T4SSs consist of Ϸ8-15 proteins, 12 of which (named VirB1-VirB11 and VirD4 according to the nomenclature of the model T4SS from A. tumefaciens) are required for secretion (3, 4). However, very little is known about how these proteins assemble to form the translocation apparatus, and about the functions of each component within the machinery. VirB2 is the major component of the A. tumefaciens pilus, and it was shown to undergo cyclization before its incorporation into extracellular pili (7,8). VirB5 and VirB7 are also associated with the pilus but appear to be minor components (9, 10). The core channel structure is likely formed by . Three putative NTPases, VirB4, VirB11, and VirD4, power the secretion apparatus, and VirB4 and VirB11 are also essential for pilus biogenesis (15-18).Recently, the structures of a VirB11 ATPase ortholog from H. pylori, HP0525 (19,20), and of a VirD4 ATPase ortholog from the conjugative plasmid R388, TrwB, have been determined (21). Both ATPases are hexameric assemblies. TrwB is architecturally related to hexamer...
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