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(1178-1222)(Mm) peptide proved that helix I
readily reaches a conformation very similar to the corresponding helix
in hSos 1, 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(1178-1222)(Mm) 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.