Structural changes induced in p21Ras upon GAP-334 complexation as probed by ESEEM spectroscopy and molecular-dynamics simulation.
BACKGROUND
The means by which the protein GAP accelerates GTP hydrolysis, and thereby downregulates growth signaling by p21Ras, is of considerable interest, particularly inasmuch as p21 mutants are implicated in a number of human cancers. A GAP "arginine finger," identified by X-ray crystallography, has been suggested as playing the principal role in the GTP hydrolysis. Mutagenesis studies, however, have shown that the arginine can only partially account for the 10(5)-fold increase in the GAP-accelerated GTPase rate of p21.


RESULTS
We report electron spin-echo envelope modulation (ESEEM) studies of GAP-334 complexed with GMPPNP bound p21 in frozen solution, together with molecular-dynamics simulations. Our results indicate that, in solution, the association of GAP-334 with GTP bound p21 induces a conformational change near the metal ion active site of p21. This change significantly reduces the distances from the amide groups of p21 glycine residues 60 and 13 to the divalent metal ion.


CONCLUSIONS
The movement of glycine residues 60 and 13 upon the binding of GAP-334 in solution provides a physical basis to interpret prior mutagenesis studies, which indicated that Gly-60 and Gly-13 of p21 play important roles in the GAP-dependent GTPase reaction. Gly-60 and Gly-13 may play direct catalytic roles and stabilize the attacking water molecule and beta,gamma-bridging oxygen, respectively, in p21. The amide proton of Gly-60 could also play an indirect role in catalysis by supplying a crucial hydrogen bonding interaction that stabilizes loop L4 and therefore the position of other important catalytic residues.
Farrar
C T
CT
Department of Chemistry, Harvard University, Cambridge, Massachusetts 02138, USA.
Ma
J
J
Singel
D J
DJ
Halkides
C J
CJ
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D005583:Fourier Analysis; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015689:Oncogene Protein p21(ras) / Q000737:chemistry* / Q000235:genetics; D012996:Solutions; D013816:Thermodynamics; D020729:p120 GTPase Activating Protein / Q000737:chemistry*
D046911:Macromolecular Substances; D012996:Solutions; D020729:p120 GTPase Activating Protein; D015689:Oncogene Protein p21(ras); D005998:Glycine