Laurent Terradot

Helicobacter pylori Type IV Secretion Apparatus Exploits β1 Integrin in a Novel RGD-Independent Manner

Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (T4SS) into host cells is a major risk factor for severe gastric diseases, including gastric cancer. However, the mechanism of translocation and the requirements from the host cell for that event are not well understood. The T4SS consists of inner- and outer membrane-spanning Cag protein complexes and a surface-located pilus. Previously an arginine-glycine-aspartate (RGD)-dependent typical integrin/ligand type interaction of CagL with α5β1 integrin was reported to be essential for CagA translocation. Here we report a specific binding of the T4SS-pilus-associated components CagY and the effector protein CagA to the host cell β1 Integrin receptor. Surface plasmon resonance measurements revealed that CagA binding to α5β1 integrin is rather strong (dissociation constant, KD of 0.15 nM), in comparison to the reported RGD-dependent integrin/fibronectin interaction (KD of 15 nM). For CagA translocation the extracellular part of the β1 integrin subunit is necessary, but not its cytoplasmic domain, nor downstream signalling via integrin-linked kinase. A set of β1 integrin-specific monoclonal antibodies directed against various defined β1 integrin epitopes, such as the PSI, the I-like, the EGF or the β-tail domain, were unable to interfere with CagA translocation. However, a specific antibody (9EG7), which stabilises the open active conformation of β1 integrin heterodimers, efficiently blocked CagA translocation. Our data support a novel model in which the cag-T4SS exploits the β1 integrin receptor by an RGD-independent interaction that involves a conformational switch from the open (extended) to the closed (bent) conformation, to initiate effector protein translocation.

The Structure of the Helicobacter Pylori Ferric Uptake Regulator Fur Reveals Three Functional Metal Binding Sites.

Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S‐C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur‐like proteins, the crystal structure of HpFur reveals a unique structured N‐terminal extension and an unusual C‐terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS4 site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site‐directed mutagenesis and spectroscopy analyses of purified wild‐type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

Architecture of the Helicobacter pylori Cag‐type IV secretion system

Type IV secretion systems (T4SS) are macromolecular assemblies used by bacteria to transport material across their membranes. T4SS are generally composed of a set of twelve proteins (VirB1–11 and VirD4). This represents a dynamic machine powered by three ATPases. T4SS are widespread in pathogenic bacteria where they are often used to deliver effectors into host cells. For example, the human pathogen Helicobacter pylori encodes a T4SS, the Cag‐T4SS, which mediates the injection of the toxin CagA. We review the progress made in the past decade in our understanding of T4SS architecture. We translate this new knowledge to derive an understanding of the structure of the H. pylori Cag system, and use recent protein–protein interaction data to refine this model.

A Sedimented Sample of a 59 kDa Dodecameric Helicase Yields High‐Resolution Solid‐State NMR Spectra

Crystal clear: Preparing solid-state NMR samples that yield high-resolution spectra displaying high sensitivity is time-consuming and complicated. A sample of the 59 kDa protein DnaB, prepared simply by preparative centrifugation, provides spectra that are as good as the ones from carefully grown microcrystals.

Structural insights into Helicobacter pylori oncoprotein CagA interaction with β1 integrin

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5β1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ∼100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer β-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1–4), which represent putative hot-spots for protein–protein interactions. The proximal part of the single-layer β-sheet, covering CSP4, is involved in specific binding of CagA to the β1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind β1 integrin to mediate its own translocation.

Structural basis of 5-nitroimidazole antibiotic resistance: the crystal structure of NimA from Deinococcus radiodurans.

5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 Å resolution. We have shown that drNimA is a homodimer in which each monomer adopts a β-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics.

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Bacterial DNA replication requires DnaA, an AAA+ ATPase that initiates replication at a specific chromosome region, oriC, and is regulated by species-specific regulators that directly bind DnaA. HobA is a DnaA binding protein, recently identified as an essential regulator of DNA replication in Helicobacter pylori. We report the crystal structure of HobA in complex with domains I and II of DnaA (DnaAI–II) from H. pylori, the first structure of DnaA bound to one of its regulators. Biochemical characterization of the complex formed shows that a tetramer of HobA binds four DnaAI–II molecules, and that DnaAI–II is unable to oligomerize by itself. Mutagenesis and protein–protein interaction studies demonstrate that some of the residues located at the HobA-DnaAI–II interface in the structure are necessary for complex formation. Introduction of selected mutations into H. pylori shows that the disruption of the interaction between HobA and DnaA is lethal for the bacteria. Remarkably, the DnaA binding site of HobA is conserved in DiaA from Escherichia coli, suggesting that the structure of the HobA/DnaA complex represents a model for DnaA regulation in other Gram-negative bacteria. Our data, together with those from other studies, indicate that HobA could play a crucial scaffolding role during the initiation of replication in H. pylori by organizing the first step of DnaA oligomerization and attachment to oriC.

Expression of Helicobacter pylori CagA domains by library-based construct screening

Highly pathogenic strains of Helicobacter pylori use a type IV secretion system to inject the CagA protein into human gastric cells. There, CagA associates with the inner side of the membrane and is tyrosine‐phosphorylated at EPIYA motifs by host kinases. The phosphorylation triggers a series of interactions between CagA and human proteins that result in a dramatic change of cellular morphology. Structural and functional analyses of the protein have proved difficult, due to the proteolytically sensitive nature of the recombinant protein. To circumvent these difficulties, we applied ESPRIT, a library‐based construct screening method, to generate a comprehensive set of 5′‐randomly deleted gene fragments. Screening of 18 432 constructs for soluble expression resulted in a panel of 40 clones, which were further investigated by large‐scale purification. Two constructs of approximately 25 and 33 kDa were particularly soluble and were purified to near homogeneity. CagA fragments larger than 40 kDa were prone to heavy proteolysis at the C‐terminus, with a favoured cleavage site near the first EPIYA motif. Thus, these well‐expressed recombinant constructs isolated are likely to be similar to those observed following natural proteolysis in human cells, and open the way for structural and functional studies requiring large amounts of purified material.

Structural similarity between the DnaA‐binding proteins HobA (HP1230) from Helicobacter pylori and DiaA from Escherichia coli

In prokaryotes, DNA replication is initiated by the binding of DnaA to the oriC region of the chromosome to load the primosome machinery and start a new replication round. Several proteins control these events in Escherichia coli to ensure that replication is precisely timed during the cell cycle. Here, we report the crystal structure of HobA (HP1230) at 1.7 Å, a recently discovered protein that specifically interacts with DnaA protein from Helicobacter pylori (HpDnaA). We found that the closest structural homologue of HobA is a sugar isomerase (SIS) domain containing protein, the phosphoheptose isomerase from Pseudomonas aeruginosa. Remarkably, SIS proteins share strong sequence homology with DiaA from E. coli; yet, HobA and DiaA share no sequence homology. Thus, by solving the structure of HobA, we unexpectedly discovered that HobA is a H. pylori structural homologue of DiaA. By comparing the structure of HobA to a homology model of DiaA, we identified conserved, surface‐accessible residues that could be involved in protein–protein interaction. Finally, we show that HobA specifically interacts with the N‐terminal part of HpDnaA. The structural homology between DiaA and HobA strongly supports their involvement in the replication process and these proteins could define a new structural family of replication regulators in bacteria.

Identification, structure and mode of action of a new regulator of the Helicobacter pylori HP0525 ATPase.

Helicobacter pylori is one of the worlds most successful human pathogens causing gastric ulcers and cancers. A key virulence factor of H. pylori is the Cag pathogenicity island, which encodes a type IV secretion system. HP0525 is an essential component of the Cag system and acts as an inner membrane associated ATPase. HP0525 forms double hexameric ring structures, with the C‐terminal domains (CTDs) forming a closed ring and the N‐terminal domains (NTDs) forming a dynamic, open ring. Here, the crystal structure of HP0525 in complex with a fragment of HP1451, a protein of previously unknown function, is reported. The HP1451 construct consists of two domains similar to nucleic acid‐binding domains. Two HP1451 molecules bind to the HP0525 NTDs on opposite sides of the hexamer, locking it in the closed form and forming a partial lid over the HP0525 chamber. From the structure, it is suggested that HP1451 acts as an inhibitory factor of HP0525 to regulate Cag‐mediated secretion, a suggestion confirmed by results of in vitro ATPase assay and in vivo pull‐down experiments.