Novel peptide recognized by RhoA GTPase.
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.
Drulis-Fajdasz
Dominika
D
Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland.
Jelen
Filip
F
Oleksy
Arkadiusz
A
Otlewski
Jacek
J
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020742:rhoA GTP-Binding Protein