Structural and functional analysis of a mutant Ras protein that is insensitive to nitric oxide activation.
Ras proteins cycle between active, guanosine triphosphate (GTP)-bound and inactive, guanosine diphospate (GDP)-bound states to mediate signal transduction pathways that promote cell growth and differentiation. It is believed that the major physiological mechanism for Ras activation is via interaction with guanine-nucleotide exchange factors (GEFs). This interaction is highly regulated and results in elevated levels of Ras-GTP by facilitating GDP dissociation. Recently, a novel mechanism of Ras activation has been proposed, whereby nitric oxide (NO) modification of Cys-118, like GEF interaction, populates Ras in its biologically active form by stimulating GDP release. Here, we describe characterization of a variant of Ras, C118S, that is insensitive to NO modification. We have measured the GTPase activity and the GDP dissociation rate of the C118S mutant and found them to be similar to wild-type Ras. We have also analyzed the structure of this mutant using multidimensional heteronuclear NMR methods. Analysis of chemical shifts and distance restraints demonstrates that this mutation has not disrupted the structure of the protein. These results suggest that NO modification of Cys-118 may not alter Ras structure and that the basis of Ras activation by NO is destabilization of a crucial interaction between residues in the GDP-binding pocket and the nucleotide. We have also found that this mutant is a more stable form of Ras at concentrations required for NMR studies, probably due to the removal of a surface-accessible cysteine residue. This stable variant may facilitate structural and biochemical investigations of Ras and other guanine-nucleotide-binding proteins containing a cysteine at this position.
Mott
H R
HR
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599, USA.
Carpenter
J W
JW
Campbell
S L
SL
D020558:GTP Phosphohydrolases / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009569:Nitric Oxide / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D010710:Phosphates; D006153:Guanosine Diphosphate; D009569:Nitric Oxide; D006147:Guanine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D018631:ras Proteins