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A Helical RGD Motif Promoting Cell Adhesion: Crystal Structures of the Helicobacter Pylori Type IV Secretion System Pilus Protein Cagl

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A Helical RGD Motif Promoting Cell Adhesion: Crystal Structures of the Helicobacter Pylori Type IV Secretion System Pilus Protein Cagl Structure Article A Helical RGD Motif Promoting Cell Adhesion: Crystal Structures of the Helicobacter pylori Type IV Secretion System Pilus Protein CagL Stephan Barden,1 Stefanie Lange,1 Nicole Tegtmeyer,3,4 Jens Conradi,2 Norbert Sewald,2 Steffen Backert,3,4 and Hartmut H. Niemann1,* 1Structural Biochemistry, Department of Chemistry, Bielefeld University, 33501 Bielefeld, Germany 2Organic and Bioorganic Chemistry, Department of Chemistry, Bielefeld University, 33501 Bielefeld, Germany 3Institute of Medical Microbiology, OvG University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany 4Department Biology, FAU University Erlangen-Nuremberg, D-91058 Erlangen, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.str.2013.08.018 SUMMARY ploits integrin receptors via a type IV secretion system (T4SS; Kwok et al., 2007). RGD tripeptide motifs frequently mediate ligand T4SSs are macromolecular assemblies that transport proteins binding to integrins. The type IV secretion system or protein-DNA complexes across the bacterial envelope, result- (T4SS) protein CagL of the gastric pathogen Helico- ing in secretion into the medium or transfer into bacterial or eu- bacter pylori also contains an RGD motif. CagL karyotic target cells (Alvarez-Martinez and Christie, 2009; decorates the T4SS pilus and may function as an Fronzes et al., 2009). T4SSs consist of three interlinked parts: adhesin for host cells. Whether CagL binds integrins a cytoplasmic/inner membrane-associated complex composed of three NTPases providing the energy for transport; a channel via its RGD motif is under debate. Here, we present spanning the bacterial envelope, also known as the core com- crystal structures of CagL revealing an elongated plex; and an external pilus. The Agrobacterium tumefaciens T- four-helix bundle that appears evolutionarily unre- DNA transfer system is the prototypic T4SS. It consists of 11 lated to the proposed VirB5 orthologs. The RGD essential VirB proteins (VirB1-VirB11) and VirD4. The virulence- motif is surface-exposed but located within a long associated T4SS of H. pylori is encoded in an 40 kb genomic a helix. This is unprecedented as previously charac- insertion, the cytotoxin-associated gene pathogenicity island terized integrin-binding RGD motifs are located (cagPAI) (Odenbreit et al., 2000). Homologs for most of the 12 within extended or flexible loops. Yet, adhesion of proteins of the A. tumefaciens VirB/D T4SS have been identified gastric epithelial cells to CagL was strictly RGD- among the 27 proteins encoded in the cagPAI, although some dependent. Comparison of seven crystallographi- are poorly defined (Buhrdorf et al., 2003; Covacci et al., 1997). cally independent molecules reveals substantial Little is known about most remaining accessory proteins en- coded by the cagPAI that lack homology to VirB/D proteins (Cen- structural flexibility. Intramolecular disulfide bonds dron and Zanotti, 2011). engineered to reduce CagL flexibility resulted in CagL is one such cagPAI protein that lacks clear sequence more stable protein, but unable to support cell adhe- similarity to any VirB/D protein. CagL localizes to the surface sion. CagL may thus partly unfold during receptor of the T4SS pilus (Kwok et al., 2007) and a DcagL mutant fails binding. to translocate the oncogenic effector protein CagA and induce interleukin-8 (IL-8) secretion in host cells (Fischer et al., 2001). Despite a mere 15% sequence identity, CagL was suggested INTRODUCTION to be an ortholog of VirB5, the prototypical adhesin of the VirB/D system primarily based on its function, size (220 amino Adhesion of animal cells to the extracellular matrix (ECM) is a acids), isoelectric point, genomic organization, and three- fundamental process in development, tissue homeostasis, and dimensional modeling (Backert et al., 2008). CagL was alterna- disease. Specific cell surface receptors including the heterodi- tively suggested to be a H. pylori-specific protein that shares meric integrins mediate interactions with ECM proteins including sequence similarity with CagH (Shaffer et al., 2011). CagL can fibronectin (Fn; Hynes, 2002; Singh et al., 2010). Fn alone is suf- be copurified with CagH and CagI, two cagPAI proteins en- ficient to induce spreading of various mammalian cell types. The coded within the same operon as CagL (Pham et al., 2012; Arg-Gly-Asp (RGD) tripeptide motif of Fn is crucial for integrin Shaffer et al., 2011). All three share a conserved C-terminal binding and RGD motifs are also present in other integrin ligands ‘‘S/T-K-I/V-I-V-K’’-hexapeptide and influence pilus formation (Leiss et al., 2008; Ruoslahti, 1996). Some bacterial and viral (Shaffer et al., 2011). pathogens target integrins, facilitating extracellular persistence CagL is the only cagPAI protein with an RGD motif and or entry into host cells (Hauck et al., 2006; Stewart and Nem- may function as a T4SS adhesin that binds to the host inte- erow, 2007; Ulanova et al., 2009). One example is the gastric grins a5b1, aVb5, and aVb3(Conradi et al., 2012a; Jime´ nez- pathogen and group 1 carcinogen Helicobacter pylori, which ex- Soto et al., 2009; Kwok et al., 2007; Wiedemann et al., 2012), Structure 21, 1931–1941, November 5, 2013 ª2013 Elsevier Ltd All rights reserved 1931 Structure Structure of HP CagL Reveals a Helical RGD Motif Table 1. CagL Proteins Used in This Study RESULTS Protein Mutation(s)/Modification Crystallization and Structure Determination of CagL Wild-Type Variants The structure of CagL (amino acids 21–237) was determined in C-HIS CagL C-term. Leu-Glu-His6 two crystal forms. To reduce surface entropy and aid crystalliza- wt CagL N-term. TEV-cleavable His6 tion (Derewenda, 2004), we replaced potentially surface CagLmeth reductive methylation of CagLwt exposed, high entropy amino acids by smaller, low entropy res- N-Terminal Deletion idues to generate the variant CagLKKQEK (Table 1). Functionality KKQEK CagLDN deletion of aa 21–61 of CagL in the T4SS was assessed by genetic complemen- Surface Entropy Reduction Variants tation of CagL in H. pylori and has been proven to be as active as CagLwt (Figure S1 available online). The structure of CagLKKQEK CagLKK69AT K69A and K70T was solved using seleno-methionine (SeMet) derivatized protein CagLKKQEK K69A, K70T, Q147A, E148T, and K149A by multi-wavelength anomalous dispersion (MAD; Table 2). Cu- RGD Variants bic crystals contained one molecule per asymmetric unit. R76A CagL R76A Following reductive methylation of primary amines of wild-type CagLR76K R76K CagL (CagLmeth), we obtained a second, tetragonal crystal CagLG77A G77A form with six molecules in the asymmetric unit. The structure KKQEK CagLD78A D78A was solved by molecular replacement (MR) using CagL Structure-Function Correlation Variants as search model. Data processing and refinement statistics for both crystal forms are provided in Table 3. While the six crystal- CagLI73N I73N lographically independent molecules of CagLmeth are similar to CagLAL80QA A80Q and L81A each other (Table S1), there are major differences to CagLKKQEK. EA87AE CagL E87A and A88E We will use the best defined molecule A of CagLmeth as a refer- G169K CagL G169K ence, and describe important differences for the higher resolu- CagLAS171SN A171S and S172N tion structure of CagLKKQEK where appropriate. C-Terminal Variants CagLC128S C128S CagL Forms an Elongated Helical Bundle ˚ ˚ CagLY225A Y225A CagL is rod-shaped with dimensions of about 90 A by 30 A and D consists of six a helices. Four long a helices (a1, a2, a5, and a6) CagL C deletion of aa 227–237 run roughly parallel to the long axis with two short perpendicular Cysteine Variants helices (a3 and a4) winding around the N-terminal part of a5(Fig- C1 CagL A71C and S172C ures 1 and S1). We will refer to CagL with the long axis oriented C2 CagL N85C and A162C vertically such that both N and C termini point up, positioning the CagLC1+C2 A71C, N85C, S172C, and A162C short helices a3 and a4 at the top. The connections between a1 Substitution and deletion mutants are derived from CagLwt covering aa and a2 and between a5 and a6 form the bottom of the molecule. 21–237. Protein intended for crystallization was TEV-cleaved. Viewed from the top, a1, a2, a5, and a6 are arranged counter- clockwise. The elongated shape and mainly a helical structure of CagL are consistent with the molecular mass estimate of but the RGD dependence of various CagL functions remains 50 kDa from analytical gel filtration (calculated mass from controversial. One group reported RGD-independent IL-8 sequence 25 kDa) and with circular dichroism (CD) spectra of induction and CagA translocation (Jime´ nez-Soto et al., 2009). CagL, respectively (Figures S2A and S3A; Conradi et al., A second group reported RGD motif-independent induction 2012b; Kwok et al., 2007). of the gastrin promoter by CagL (Wiedemann et al., 2012). Although the fold is clearly defined, CagL is characterized by Others found CagA translocation (Kwok et al., 2007), IL-8 in- substantial structural flexibility. The most invariant part of CagL duction (Gorrell et al., 2012), host cell Src and FAK kinase is a three-helix bundle formed by the upper half of a2, a5, and activation (Kwok et al., 2007; Tegtmeyer et al., 2010), and a6 together with a3(Figures 1A and 1B). This CagL core is spreading of various cells on immobilized CagL (Tegtmeyer held together by a cluster of aromatic residues: Phe86, Phe92, et al., 2010) to be RGD motif-dependent. The importance of Phe93, and Phe100 from a2, Tyr163 from a5, and His200, the RGD motif is further underlined by synthetic RGD peptides Phe204, and Tyr207 from a6. Minor structural variations are that interfere with cell adhesion to wild-type CagL (Conradi found in the C terminus of a5, the N terminus of a6, and the con- et al., 2012a) and complement deficiencies of T4SS pilus as- necting short loop. This slightly more flexible region begins with a sembly and CagA phosphorylation in a DcagL H.
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