Preferences help
enabled [disable] Abstract
Number of results
2006 | 53 | 3 | 515-524
Article title

Novel peptide recognized by RhoA GTPase

Title variants
Languages of publication
A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA - R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+- and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.
Physical description
  • Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland
  • Arkin MR, Wells JA (2004) Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat Rev Drug Discov 3: 301-317.
  • Bar-Sagi D, Hall A (2000) Ras, Rho GTPases: a family reunion. Cell 103: 227-238.
  • Benitah SA, Valeron PF, van Aelst L, Marshall CJ, Lacal JC (2004) Rho GTPases in human cancer: an unresolved link to upstream, downstream transcriptional regulation. Biochim Biophys Acta 1705: 121-132.
  • Burke CJ, Volkin DB, Mach H, Middaugh CR (1993) Effect of polyanions on the unfolding of acidic fibroblast growth factor. Biochemistry 32: 6419-6426.
  • Cochran AG (2001) Protein-protein interfaces: mimics, inhibitors. Curr Opin Chem Biol 5: 654-659.
  • Corbett KD, Alber T (2001) The many faces of Ras: recognition of small GTP-binding proteins. Trends Biochem Sci 26: 710-716.
  • Derewenda U, Oleksy A, Stevenson AS, Korczynska J, Dauter Z, Somlyo AP, Otlewski J, Somlyo AV, Derewenda ZS (2004) The crystal structure of RhoA in complex with the DH/PH fragment of PDZRhoGEF, an activator of the Ca(2+) sensitization pathway in smooth muscle. Structure (Camb) 12: 1955-1965.
  • Deshayes K, Schaffer ML, Skelton NJ, Nakamura GR, Kadkhodayan S, Sidhu SS (2002) Rapid identification of small binding motifs with high-throughput phage display: discovery of peptidic antagonists of IGF-1 function. Chem Biol 9: 495-505.
  • Dvorsky R, Ahmadian MR (2004) Always look on the bright site of Rho: structural implications for a conserved intermolecular interface. EMBO Rep 5: 1130-1136.
  • Fack F, Hugle-Dorr B, Song D, Queitsch I, Petersen G, Bautz EK (1997) Epitope mapping by phage display: random versus gene-fragment libraries. J Immunol Methods 206: 43-52.
  • Fairbrother WJ, Christinger HW, Cochran AG, Fuh G, Keenan CJ, Quan C, Shriver SK, Tom JY, Wells JA, Cunningham BC (1998) Novel peptides selected to bind vascular endothelial growth factor target the receptor-binding site. Biochemistry 37: 17754-17764.
  • Gao Y, Dickerson JB, Guo F, Zheng J, Zheng Y (2004) Rational design, characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA 101: 7618-7623.
  • Hildebrand JD, Taylor JM, Parsons JT (1996) An SH3 domain-containing GTPase-activating protein for Rho, Cdc42 associates with focal adhesion kinase. Mol Cell Biol 16: 3169-3178.
  • Jelen F, Oleksy A, Smietana K, Otlewski J (2003) PDZ domains - common players in the cell signaling. Acta Biochim Polon 50: 985-1017.
  • Kapust RB, Tozser J, Fox JD, Anderson DE, Cherry S, Copeland TD, Waugh DS (2001) Tobacco etch virus protease: mechanism of autolysis, rational design of stable mutants with wild-type catalytic proficiency. Protein Eng 14: 993-1000.
  • Karnoub AE, Symons M, Campbell SL, Der CJ (2004) Molecular basis for Rho GTPase signaling specificity. Breast Cancer Res Treat 84: 61-71.
  • Kato-Stankiewicz J, Hakimi I, Zhi G, Zhang J, Serebriiskii I, Guo L, Edamatsu H, Koide H, Menon S, Eckl R, Sakamuri S, Lu Y, Chen QZ, Agarwal S, Baumbach WR, Golemis EA, Tamanoi F, Khazak V (2002) Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells. Proc Natl Acad Sci USA 99: 14398-14403.
  • Ladner RC, Sato AK, Gorzelany J, de Souza M (2004) Phage display-derived peptides as therapeutic alternatives to antibodies. Drug Discov Today 9: 525-529.
  • Longenecker KL, Zhang B, Derewenda U, Sheffield PJ, Dauter Z, Parsons JT, Zheng Y, Derewenda ZS (2000) Structure of the BH domain from graf, its implications for Rho GTPase recognition. J Biol Chem 275: 38605-38610.
  • Longenecker K, Read P, Lin SK, Somlyo AP, Nakamoto RK, Derewenda ZS (2003) Structure of a constitutively activated RhoA mutant (Q63L) at 1.55 A resolution. Acta Crystallogr D Biol Crystallogr 59: 876-880.
  • Meiering EM, Bycroft M, Fersht AR (1991) Characterization of phosphate binding in the active site of barnase by site-directed mutagenesis, NMR. Biochemistry 30: 11348-11356.
  • Melisi D, Troiani T, Damiano V, Tortora G, Ciardiello F (2004) Therapeutic integration of signal transduction targeting agents, conventional anti-cancer treatments. Endocr Relat Cancer 11: 51-68.
  • Murase K, Morrison KL, Tam PY, Stafford RL, Jurnak F, Weiss GA (2003) EF-Tu binding peptides identified, dissected, affinity optimized by phage display. Chem Biol 10: 161-168.
  • Paduch M, Jelen F, Otlewski J (2001) Structure of small G proteins and their regulators. Acta Biochim Polon 48: 829-850.
  • Sancho J, Meiering EM, Fersht AR (1991) Mapping transition states of protein unfolding by protein engineering of ligand-binding sites. J Mol Biol 221: 1007-1014.
  • Santoro MM, Bolen DW (1992) A test of the linear extrapolation of unfolding free energy changes over an extended denaturant concentration range. Biochemistry 31: 4901-4907.
  • Schmidt S, Diriong S, Mery J, Fabbrizio E, Debant A (2002) Identification of the first Rho-GEF inhibitor, TRIPalpha, which targets the RhoA-specific GEF domain of Trio. FEBS Lett 523: 35-42.
  • Sheffield P, Garrard S, Derewenda Z (1999) Overcoming expression, purification problems of RhoGDI using a family of 'parallel' expression vectors. Protein Expr Purif 15: 34-39.
  • Shimizu T, Ihara K, Maesaki R, Kuroda S, Kaibuchi K, Hakoshima T (2000) An open conformation of switch I revealed by the crystal structure of a Mg2+-free form of RHOA complexed with GDP. Implications for the GDP/GTP exchange mechanism. J Biol Chem 275: 18311-1817.
  • Sidhu SS (2000) Phage display in pharmaceutical biotechnology. Curr Opin Biotechnol 11: 610-616.
  • Sidhu SS (2001) Engineering M13 for phage display. Biomol Eng 18: 57-63.
  • Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000) Phage display for selection of novel binding peptides. Methods Enzymol 328: 333-363.
  • Sidhu SS, Fairbrother WJ, Deshayes K (2003) Exploring protein-protein interactions with phage display. Chembiochem 4: 14-25.
  • Thapar R, Karnoub AE, Campbell SL (2002) Structural, biophysical insights into the role of the insert region in Rac1 function. Biochemistry 41: 3875-3883.
  • Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate, for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci USA 89: 4884-4887.
  • Wei Y, Zhang Y, Derewenda U, Liu X, Minor W, Nakamoto RK, Somlyo AV, Somlyo AP, Derewenda ZS (1997) Crystal structure of RhoA-GDP, its functional implications. Nat Struct Biol 4: 699-703.
  • Zhang B, Wang ZX, Zheng Y (1997) Characterization of the interactions between the small GTPase Cdc42, its GTPase-activating proteins, putative effectors. Comparison of kinetic properties of Cdc42 binding to the Cdc42-interactive domains. J Biol Chem 272: 21999-22007.
  • Zhang B, Zhang Y, Wang Z, Zheng Y (2000) The role of Mg2+ cofactor in the guanine nucleotide exchange, GTP hydrolysis reactions of Rho family GTP-binding proteins. J Biol Chem 275: 25299-25307.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.