AN NMR COMPARISON OF THE CHANGES PRODUCED BY DIFFERENT GUANOSINE 5'-TRIPHOSPHATE ANALOGS IN WILD-TYPE AND ONCOGENIC MUTANT P21RAS

We have used nuclear magnetic resonance spectroscopy to compare the co nformational changes produced by replacement of bound GDP by the GTP a nalogs guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) and guanyiyl (b eta,gamma-imido)diphosphate (GMPPNP) in wild-type p21ras as well as th e oncogenic mutant (G12D)p21ras. We have used isotope-edited nuclear m agnetic resonance spectroscopy to observe the amide resonances of sele ctively [N-15]glycine and [N-15]isoleucine labeled p21ras-nucleotide c omplexes. We find that eight of the nine resonances that respond stron gly to GTPgammaS and GMPPNP binding are the same but that the nature o f the effect appears different. With GTPgammaS, seven new resonances r eplace the eight resonances specifically associated with GDP-p21ras, b ut in GMPPNP-p21ras only two resonances replace the GDP-specific reson ances that are lost. The resonance of Gly 60 is clearly shown to be re sponsive to replacement of GDP by GMPPNP, in addition to glycines 10, 12, 13, 15, and 7 5 and isoleucines 36, 21, and one other, that were f ound to respond to GTPgammaS by Miller et al. [Miller, A.-F., Papastav ros, M. Z., & Redfield, A.G. (1992) Biochemistry 31, 10208-10216). The two GMPPNP-specific resonances observed appear in positions similar t o GTP-gammaS-specific resonances, and the GTPgammaS-specific resonance s, although not lost altogether, are weaker than the GDP-specific reso nances they replace. Thus, the two GTP analogs have similar effects on the spectrum of p21ras, suggesting that the effects are due to featur es common to both analogs. We propose that active site resonance inten sities are specifically attenuated when GTP analogs are bound because interactions with the gamma-phosphate of GTP analogs couple the flexib le loops 2 and 4 to the rigid loop 1 of the active site. The conformat ional heterogeneity and dynamics of loops 2 and 4 would be constrained by loop 1 but also transmitted to it. Coupled conformational exchange on a common intermediate time scale could explain the simultaneous lo ss of resonances from all three loops in the active site. In our compa rison of wild-type and (G12D) GDP-p21ras, we find that the resonance o f Ile 36 is not visible in (G12D)p21ras. In (G12D)p21ras, replacement of GDP by GTP-gammaS causes the resonances of glycines 10, 13, 15, 60, and 75 and isoleucine 21 and four others to shift from their GDP-spec ific positions. GTPgammaS-specific resonances are observed for all but two of these. The assigned responsive resonances all correspond to re sidues in the active site or connected to it. Largely the same resonan ces respond to GMPPNP binding, but only four corresponding resonances specific to GMPPNP-(G12D)p21ras are observed. Thus, replacement of gly cine 12 by aspartate only slightly alters the responsiveness of the gr ound state of p21ras to nucleotide replacement. Furthermore, the obser ved GTPgammaS-specific resonances of (G12D)p21ras are close to GTPgamm aS-specific resonances of wild-type p21ras, even though several of the GDP-specific (G12D)p21ras resonances differ significantly from those of wild-type p21ras.