Amino Acids

, Volume 43, Issue 1, pp 219–232 | Cite as

Cyclic RGD peptides interfere with binding of the Helicobacter pylori protein CagL to integrins αVβ3 and α5β1

  • Jens Conradi
  • Sylwia Huber
  • Katharina Gaus
  • Felix Mertink
  • Soledad Royo Gracia
  • Ulf Strijowski
  • Steffen Backert
  • Norbert SewaldEmail author
Original Article


The human pathogen Helicobacter pylori that may cause different gastric diseases exploits integrins for infection of gastric cells. The H. pylori protein CagL present on the outer region of the type IV secretion pilus contains an RGD sequence (-Arg-Gly-Asp-) that enables binding to cells presenting integrins α5β1 and αVβ3. This interaction can be inhibited with conformationally designed cyclic RGD peptides derived from the CagL epitope -Ala-Leu-Arg-Gly-Asp-Leu-Ala-. The inhibition of the CagL–αVβ3 interaction by different RGD peptides strongly suggests the importance of the RGD motif for CagL binding. CagL point mutants (RAD, RGA) show decreased affinity to integrin αVβ3. Furthermore, structure–activity relationship studies with cyclic RGD peptides in a spatial screening approach show the distinct influence of the three-dimensional arrangement of RGD motif on the ability to interfere with this interaction. Resulting from these studies, similar structural requirements for the CagL epitope as previously suggested for other ligands of integrin αVβ3 are proposed.


CagL Integrins αVβ3 RGD peptides SAR studies 



Dulbecco's phosphate buffered saline






Ethylenediaminetetraacetic acid


1-[Bis-(dimethylamino)methyliumyl]-1H-1,2,3-triazolo[4,5-b]pyridine-3-oxide hexafluorophosphate


Minimum essential medium


2-(N-Morpholino)ethanesulfonic acid






3-[Bis(dimethylamino)methyliumyl]-3H-benzotriazol-1-oxide tetrafluoroborate


Trifluoroacetic acid





The authors thank Carmela Michalek and Marco Wißbrock for skillful technical assistance. Financial support came from the NRW Graduate School in Bioinformatics and Genome Research, Bielefeld University (PhD Fellowship to K.G.), the EU (Marie Curie Fellowship to S.R.G.), and Deutsche Forschungsgemeinschaft, which is gratefully acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2011_1066_MOESM1_ESM.doc (766 kb)
Supplementary material 1 (DOC 766 kb)


  1. Amieva MR, El-Omar EM (2008) Host-bacterial interactions in Helicobacter pylori infection. Gastroenterol 134:306–320CrossRefGoogle Scholar
  2. Asahi M, Azuma T, Ito S, Ito Y, Suto H, Nagai Y, Tsubokawa M, Tohyama Y, Maeda S, Omata M, Suzuki T, Sasakawa CJ (2000) Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells. Exp Med 191:593–602CrossRefGoogle Scholar
  3. Aumailley M, Gurrath M, Müller G, Calvete J, Timpl R, Kessler H (1991) Arg-Gly-Asp constrained with cyclic pentapeptides. FEBS Lett 291:50–54PubMedCrossRefGoogle Scholar
  4. Backert S, Meyer TF (2006) Type IV secretion systems and their effectors in bacterial pathogenesis. Curr Opin Microbiol 9:207–212PubMedCrossRefGoogle Scholar
  5. Backert S, Selbach M (2008) Role of type IV secretion in Helicobacter pylori pathogenesis. Cell Microbiol 10:1573–1581PubMedCrossRefGoogle Scholar
  6. Backert S, Ziska E, Brinkmann V, Zimny-Arndt U, Faucinnier A, Jungblut PR, Neumann M, Meyer TF (2000) Translocation of the Helicobacter pylori CagA protein in gastric epithelial cells by a type IV secretion apparatus. Cell Microbiol 2:155–164PubMedCrossRefGoogle Scholar
  7. Backert S, Fronzes R, Waksman G (2008) VirB2 and VirB5 proteins: specialized adhesins in bacterial type-IV secretion systems? Trends Microbiol 16:409–413PubMedCrossRefGoogle Scholar
  8. Bax A, Davis DG (1985a) Practical aspects of two-dimensional transverse NOE spectroscopy. J Magn Reson 63:207–213Google Scholar
  9. Bax A, Davis DG (1985b) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J Magn Reson 65:355–360Google Scholar
  10. Bax A, Summers MFJ (1986) 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J Am Chem Soc 108:2093–2094CrossRefGoogle Scholar
  11. Bourzac KM, Guillemin K (2005) Helicobacter pylori–host cell interactions mediated by type IV secretion. Cell Microbiol 7:911–919PubMedCrossRefGoogle Scholar
  12. Cascales E, Christie PJ (2003) The versatile bacterial type IV secretion systems. Nat Rev Microbiol 1:137–149PubMedCrossRefGoogle Scholar
  13. Charo IF, Nannizzi L, Smith JW, Cheresh DA (1990) The vitronectin receptor αVβ3 binds fibronectin and acts in concert with α5β1 in promoting cellular attachment and spreading on fibronectin. J Cell Biol 111:2795–2800PubMedCrossRefGoogle Scholar
  14. Dechantsreiter MA, Planker E, Mathä B, Lohof E, Hölzemann G, Jonczyk A, Goodman SL, Kessler H (1999) N-methylated cyclic RGD peptides as highly active and selective αVβ3 integrin antagonists. J Med Chem 42:3033–3040PubMedCrossRefGoogle Scholar
  15. Eble JA, Kühn K (eds) (1997) Integrin-ligand interaction. Springer, HeidelbergGoogle Scholar
  16. Geerke DP, Oostenbrink C, van der Vegt NFA, van Gunsteren WF (2004) An effective force field for molecular dynamics simulations of dimethyl sulfoxide and dimethyl sulfoxide-water mixtures. J Phys Chem B 108:1436–1445CrossRefGoogle Scholar
  17. Goddard TD, Kneller DG, Sparky 3, University of California, San FranciscoGoogle Scholar
  18. Gottschalk K-E, Kessler H (2002) The structures of integrins and integrin-ligand complexes: implications for drug design and signal transduction. Angew Chem Int Ed 41:3767–3774CrossRefGoogle Scholar
  19. Guthöhrlein EW, Malešević M, Majer Z, Sewald N (2007) Secondary structure inducing potential of ß-amino acids: torsion angle clustering facilitates comparison and analysis of the conformation during MD trajectories. Biopolymers 88:829–839PubMedCrossRefGoogle Scholar
  20. Haubner R, Finsinger D, Kessler H (1997) Stereoisomeric peptide libraries and peptidomimetics for designing selective inhibitors of the αVβ3 integrin for a new cancer therapy. Angew Chem Int Ed 36:1374–1389CrossRefGoogle Scholar
  21. Hoffmann C, Ohlsen K, Hauck CR (2011) Integrin-mediated uptake of fibronectin-binding bacteria. Eur J Cell Biol. doi: 10.1016/j.ejcb.2011.03.001
  22. Isberg RR, Barnes PJ (2001) Subversion of integrins by enteropathogenic Yersinia. Cell Sci 114:21–28Google Scholar
  23. Isberg RR, Leong JM (1990) Multiple β1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 60:861–871PubMedCrossRefGoogle Scholar
  24. Isberg RR, Tran Van Nhieu G (1994) Binding and internalization of microorganisms by integrin receptors. Trends Microbiol 2:10–14PubMedCrossRefGoogle Scholar
  25. Jackson T, Sheppard D, Denyer M, Blakemore W, King AMQ (2000) The epithelial integrin αVβ6 is a receptor for foot-and-mouth disease virus. J Virol 74:4949–4956PubMedCrossRefGoogle Scholar
  26. Jackson T, Mould AP, Sheppard D, King AMQ (2002) Integrin αVβ1 is a receptor for foot-and-mouth disease virus. J Virol 76:935–941PubMedCrossRefGoogle Scholar
  27. Jiménez-Soto LF, Kuttner S, Sewald X, Ertl C, Weiss E, Kapp U, Rohde M, Pirch T, Jung K, Retta SF, Terradot L, Fischer W, Haas R (2009) Helicobacter pylori type IV secretion apparatus exploits β1 integrin in a novel RGD-independent manner. PLoS Pathog 5(12):e1000684PubMedCrossRefGoogle Scholar
  28. Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  29. Kwok T, Backert S, Schwarz H, Berger J, Meyer TF (2002) Specific Entry of Helicobacter pylori into cultured gastric epithelial cells via a zipper-like mechanism. Infect Immun 70:2108–2120PubMedCrossRefGoogle Scholar
  30. Kwok T, Zabler D, Urman S, Rohde M, Hartig R, Wessler S, Misselwitz R, Berger J, Sewald N, König W, Backert S (2007) Helicobacter exploits integrin for type IV secretion and kinase activation. Nature 449:862–866PubMedCrossRefGoogle Scholar
  31. Lohof E, Planker E, Mang C, Burkhart F, Dechantsreiter MA, Haubner R, Wester H-J, Schwaiger M, Hölzemann G, Goodmann SL, Kessler H (2000) Carbohydrate derivatives for use in drug design:cyclic αV-selective RGD peptides. Angew Chem Int Ed 39:2761–2764CrossRefGoogle Scholar
  32. Macura S, Huang Y, Suter D, Ernst RR (1981) Two-dimensional chemical exchange and cross-relaxation spectroscopy of coupled nuclear spins. J Magn Reson 43:259–281Google Scholar
  33. Malešević M, Strijowski U, Bächle D, Sewald N (2004) An improved method for the solution cyclization of peptides under pseudo-high dilution conditions. J Biotechnol 112:73–77PubMedCrossRefGoogle Scholar
  34. Markley JL, Bax A, Arata Y, Hilbers CW, Kaptein R, Sykes BD, Wright PE, Wüthrich K (1998) Recommendations for the presentation of NMR structures of proteins and nucleic acids. J Biomol NMR 12:1–23PubMedCrossRefGoogle Scholar
  35. Mas-Moruno C, Rechenmacher F, Kessler H (2010) Cilengitide:the first anti-angiogenic small molecule drug candidate design, synthesis and clinical evaluation. Anti Canc Agents Med Chem 10:753–768CrossRefGoogle Scholar
  36. Meyer A, Auernheimer J, Modlinger A, Kessler H (2006) Targeting RGD recognizing integrins: drug development, biomaterial research, tumor imaging and targeting. Curr Pharm Des 12:2723–2747PubMedCrossRefGoogle Scholar
  37. Müller G, Gurrath M, Kessler H (1994) Pharmacophore refinement of gpIIb/IIIa antagonists based on comparative studies of antiadhesive cyclic and acyclic RGD peptides. J Comp Aided Mol Des 8:709–730CrossRefGoogle Scholar
  38. Pierschbacher MD, Hayman EG, Ruoslahti E (1981) Location of the cell-attachment site in fibronectin with monoclonal antibodies and proteolytic fragments of the molecule. Cell 26:259–267PubMedCrossRefGoogle Scholar
  39. Rance M, Sørensen OW, Bodenhausen G, Wagner G, Ernst RR, Wüthrich K (1983) Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun 117:479–485PubMedCrossRefGoogle Scholar
  40. Rieder G, Fischer W, Haas R (2005) Interaction of Helicobacter pylori with host cells: function of secreted and translocated molecules. Curr Opin Microbiol 8:67–73PubMedCrossRefGoogle Scholar
  41. Royo Gracia S, Gaus K, Sewald N (2009) Synthesis of chemically modified bioactive peptides: recent advances, challenges and developments for medicinal chemistry. Future Med Chem 1:1289–1310CrossRefGoogle Scholar
  42. Ruoslahti E (1996) RGD and other recognition sequences for integrins. Annu Rev Cell Dev Biol 12:697–715PubMedCrossRefGoogle Scholar
  43. Ruoslahti E, Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238:491–497PubMedCrossRefGoogle Scholar
  44. Schuler LD, Daura X, van Gunsteren WF (2001) An improved GROMOS 96 force field for aliphatic hydrocarbons in the condensed phase. J Comput Chem 22:1205–1218CrossRefGoogle Scholar
  45. Schumann F, Müller A, Koksch M, Müller G, Sewald N (2000) Are β-amino acids γ-turn mimetics?—exploring a new design principle for bioactive cyclopeptides. J Am Chem Soc 122:12009–12010CrossRefGoogle Scholar
  46. Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Res 160:65–73CrossRefGoogle Scholar
  47. Serini G, Valdembri D, Bussolino F (2006) Integrins and angiogenesis: a sticky business. Exp Cell Res 312:651–658PubMedCrossRefGoogle Scholar
  48. Shaka A, Lee CJ, Davis DG (1988) Iterative schemes for bilinear operators; application to spin decoupling. J Magn Reson 77:274–293Google Scholar
  49. Sheppard D (2003) Functions of pulmonary epithelial integrins: from development to disease. Physiol Rev 83:673–686PubMedGoogle Scholar
  50. Stewart PL, Nemerow GR (2007) Cell integrins: commonly used receptors for diverse viral pathogens. Trends Microbiol 15:500–507PubMedCrossRefGoogle Scholar
  51. Tegtmeyer N, Hartig R, Delahay RM, Rohde M, Brandt S, Conradi J, Takahashi S, Smolka AJ, Sewald N, Backert S (2010) A small fibronectin-mimicking protein from bacteria induces cell spreading and focal adhesion formation. J Biol Chem 285:23515–23526PubMedCrossRefGoogle Scholar
  52. Urman S, Gaus K, Yang Y, Strijowski U, De Pol S, Reiser O, Sewald N (2007) β-ACC containing RGD peptides efficiently inhibit tumor cell adhesion. Angew Chem Int Ed 46:3976–3978CrossRefGoogle Scholar
  53. Weide T, Modlinger A, Kessler H (2007) Spatial screening for the identification of the bioactive conformation of integrin ligands. Top Curr Chem 272:1–50CrossRefGoogle Scholar
  54. Wickham TJ, Filardo EJ, Cheresh DA, Nemerow GR (1994) Integrin αVβ5 selectively promotes adenovirus mediated cell membrane permeabilization. J Cell Biol 127:257–264PubMedCrossRefGoogle Scholar
  55. Xiong J-P, Stehle T, Zhang R, Joachimiak A, Frech M, Goodman SL, Arnaout MA (2002) Crystal structure of the extracellular segment of integrin alpha V beta3 in complex with an Arg-Gly-Asp ligand. Science 296:151–155PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Jens Conradi
    • 1
  • Sylwia Huber
    • 1
    • 4
  • Katharina Gaus
    • 1
    • 3
  • Felix Mertink
    • 1
  • Soledad Royo Gracia
    • 1
    • 6
  • Ulf Strijowski
    • 1
    • 5
  • Steffen Backert
    • 2
  • Norbert Sewald
    • 1
    Email author
  1. 1.Department of ChemistryBielefeld UniversityBielefeldGermany
  2. 2.School of Biomolecular and Biomedical SciencesUniversity College DublinDublinIreland
  3. 3.SyngentaSteinSwitzerland
  4. 4.F. Hoffmann-La RocheBaselSwitzerland
  5. 5.German Institute of Food TechnologiesQuakenbrückGermany
  6. 6.Institute for Research in BiomedicineBarcelonaSpain

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