Structure determination and dynamics of peptides overlapping the catalytic hairpin of the Ras-specific GEF Cdc25(Mm).
Ras proteins are small G proteins playing a major role in eukaryotic signal transduction. Guanine nucleotide exchange factors (GEF) stimulate GDP/GTP exchange, resulting in the formation of the active Ras-GTP complex. In mammalian cells, two major Ras-specific GEF exist: Sos-like and Cdc25-like. To date, structural data are available only for Cdc25(Mm). We designed and synthesized Cdc25(Mm)-derived peptides spanning residues corresponding to the hSos1 HI helical hairpin that has been implicated in the GEF catalytic mechanism. NMR experiments on a chemically synthesized Cdc25(Mm)(1178-1222) peptide proved that helix I readily reaches a conformation very similar to the corresponding helix in hSos1, while residues corresponding to helix H in hSos1 show higher conformational flexibility. Molecular dynamics studies with the appropriate solvent model showed that different conformational spaces are available for the peptide. Since helix H is making several contacts with Ras and a Cdc25(Mm)(1178-1222) peptide is able to bind nucleotide-free Ras in a BIAcore assay, the peptide must be able to obtain the proper Ras-interacting conformation, at least transiently. These results indicate that rational design and improvement of the Ras-interacting peptides should take into account conformational and flexibility features to obtain molecules with the appropriate biochemical properties.
Consonni
Roberto
R
Istituto per lo Studio delle Macromolecole, Lab NMR, CNR, v. Ampere 56, I-20131 Milan, Italy. roberto.consonni@ismac.cnr.it
Arosio
Ivana
I
Recca
Teresa
T
Longhi
Renato
R
Colombo
Giorgio
G
Vanoni
Marco
M
D000595:Amino Acid Sequence; D002384:Catalysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D020666:ras-GRF1 / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D020666:ras-GRF1