Adhesion of several cell lines to Helicobacter pylori CagL is mediated by integrin αVß6 via an RGDLXXL motif.

The Helicobacter pylori type IV secretion system pilus protein CagL mediates interaction with host cells via its RGD motif. Here, we analyzed prerequisites for this interaction within CagL and on host cells. Various human cell lines were tested for adhesion to CagL. HT-29 and 23132/87 cells adhered to immobilized recombinant CagL in an RGD-dependent manner, while 293T (human embryonic kidney) and A549 cells did not. In a competitive ELISA, CagL competed with fibronectin for binding to the ectodomains of integrins αVß6 and αVß8 but not of αVß1, αVß3, αVß5 and α5ß1. Integrin αVß6 acts as receptor for several viruses exposing an RGDLXXL motif. CagL also contains an RGDLXXL sequence. We individually mutated Leu79 and Leu82 of this motif to threonine, although both leucines are buried in the hydrophobic core. Surprisingly, the ability of CagL variants L79T and L82T to support adhesion was significantly reduced for 23132/87 cells and lost for MKN-45 and HT-29 cells. The role of integrin αVß6 in adhesion to CagL was investigated using SW480 cells transfected with the integrin ß6 subunit (SW480ß6). These cells adhered to CagL in an RGD-dependent manner, while mock-transfected SW480 cells did not. The antibody 3G9 that blocks the function of integrin αVß6 inhibited adhesion of SW480ß6, MKN-45, 23132/87 and HT-29 cells to CagL. In summary, CagL features an RGDLXXL motif facilitating adhesion of several human cell lines via integrin αVß6. The buried location of Leu79 and Leu82 supports our previously published hypothesis that CagL partly unfolds upon integrin binding.

Structure of a three-dimensional domain-swapped dimer of the Helicobacter pylori type IV secretion system pilus protein CagL.

A new crystal form of the Helicobacter pylori type IV secretion system (T4SS) pilus protein CagL is described here. In contrast to two previously reported monomeric structures, CagL forms a three-dimensional domain-swapped dimer. CagL dimers can arise during refolding from inclusion bodies or can form spontaneously from purified monomeric CagL in the crystallization conditions. Monomeric CagL forms a three-helix bundle, with which the N-terminal helix is only loosely associated. In the new crystal form, the N-terminal helix is missing. The domain swap is owing to exchange of the C-terminal helix between the two protomers of a dimer. A loop-to-helix transition results in a long helix of 108 amino acids comprising the penultimate and the last helix of the monomer. The RGD motif of dimeric CagL adopts an α-helical conformation. In contrast to the previously reported structures, the conserved and functionally important C-terminal hexapeptide is resolved. It extends beyond the three-helix bundle as an exposed helical appendage. This new crystal form contributes to the molecular understanding of CagL by highlighting rigid and flexible regions in the protein and by providing the first view of the C-terminus. Based on the structural features, a previously unrecognized homology between CagL and CagI is discussed.

A helical RGD motif promoting cell adhesion: crystal structures of the Helicobacter pylori type IV secretion system pilus protein CagL.

RGD tripeptide motifs frequently mediate ligand binding to integrins. The type IV secretion system (T4SS) protein CagL of the gastric pathogen Helicobacter pylori also contains an RGD motif. CagL decorates the T4SS pilus and may function as an adhesin for host cells. Whether CagL binds integrins via its RGD motif is under debate. Here, we present crystal structures of CagL revealing an elongated four-helix bundle that appears evolutionarily unrelated to the proposed VirB5 orthologs. The RGD motif is surface-exposed but located within a long α helix. This is unprecedented as previously characterized integrin-binding RGD motifs are located within extended or flexible loops. Yet, adhesion of gastric epithelial cells to CagL was strictly RGD-dependent. Comparison of seven crystallographically independent molecules reveals substantial structural flexibility. Intramolecular disulfide bonds engineered to reduce CagL flexibility resulted in more stable protein, but unable to support cell adhesion. CagL may thus partly unfold during receptor binding.

Structure of Shigella IpgB2 in complex with human RhoA: implications for the mechanism of bacterial guanine nucleotide exchange factor mimicry.

A common theme in bacterial pathogenesis is the manipulation of eukaryotic cells by targeting the cytoskeleton. This is in most cases achieved either by modifying actin, or indirectly via activation of key regulators controlling actin dynamics such as Rho-GTPases. A novel group of bacterial virulence factors termed the WXXXE family has emerged as guanine nucleotide exchange factors (GEFs) for these GTPases. The precise mechanism of nucleotide exchange, however, has remained unclear. Here we report the structure of the WXXXE-protein IpgB2 from Shigella flexneri and its complex with human RhoA. We unambiguously identify IpgB2 as a bacterial RhoA-GEF and dissect the molecular mechanism of GDP release, an essential prerequisite for GTP binding. Our observations uncover that IpgB2 induces conformational changes on RhoA mimicking DbI- but not DOCK family GEFs. We also show that dissociation of the GDP.Mg(2+) complex is preceded by the displacement of the metal ion to the alpha-phosphate of the nucleotide, diminishing its affinity to the GTPase. These data refine our understanding of the mode of action not only of WXXXE GEFs but also of mammalian GEFs of the DH/PH family.