Identification of resonances from an oncogenic activating locus of human N-RAS-encoded p21 protein using isotope-edited NMR.

Abstract

A sample ofEscherichia coil-expressed human N-RAS-encoded p21, a 21-kDa protein, was selectively labeled with '5N at each of the 14 glycine amide positions. Twodimensional proton-observe '5N correlation spectra showed one peak for each glycine residue. Five glycine resonances were identified with residues near the nucleotide binding site and provide useful reporters of several oncogene-activating positions. Three ofthese resonances were asigned to residues 10, 15, and 115 from the spectrum ofa sample that was also labeled with ['3C]valine. These resonances showed extra splitting or broadening due to the 13C label, which could be eliminated by 13Cdecoupling. Two other peaks were unambiguously identified as Gly-12 and Gly-13 using a one-dimensional edited nuclear Overhauser experiment and by spectral comparison with an Asp-12 mutant. These assignments have provided several sitespecific probes of critical domains in p21. In this report we describe methods used to identify important glycine residues in N-RAS-encoded p21 (p21), a guanine nucleotide-binding protein that plays a crucial role in control of cellular growth (6). N-RAS is one of three distinct mammalian genes (N-RAS, K-RAS, and H-RAS) that encode highly related 21-kDa proteins. These proteins are related to several other families of guanine nucleotide-binding proteins (mammalian signal-transducing proteins, elongation factor Tu). Certain mutations in the coding sequence of RAS genes give rise to p21 proteins with transforming properties, and these altered proteins have been found in many human and animal tumor cells. The structure of N-RAS p21 has been inferred by analogy with elongation factor Tu (7, 8), and a preliminary x-ray study of H-RAS-encoded p21 has appeared (9). Six glycines common to both H-RAS and N-RAS p21 are near the guanine nucleotide-binding site . Several oncogenic activating positions near this site have been genetically identified (residues 12, 13, 15, 16, 59, 116, and 119). In particular, Gly-12 is in the phosphatebinding domain. This position appears to be crucial since substitution ofany other amino acid (except proline) generates mutant p21 proteins with transformation-inducing properties.In addition to the normal N-RAS gene product, we have also studied a p21 transforming mutant that contains aspartate instead of glycine at position 12. We have used isotopeedited NMR methods to identify active site glycine resonances in ['-N]glycine p21. These resonances have been used as site-specific probes to investigate spectral variations associated with p21 point mutations or ligand changes in critical domains of p21 (S.C.B., unpublished data).The NMR methods used to obtain resolved signals will not be detailed here. As already indicated, through a combination of proton and '5N pulsed excitation we can obtain a 2D NMR spectral map that shows peaks only from the few "5NH 817The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance wi...

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D

“…Peak B in the K-Ras-GDP spectrum in Figure 3 has a shoulder, suggesting contributions from two protons, at around 10.7 ppm. A similar peak was previously assigned to the overlapped backbone amide protons of G13 and K16, respectively (Burk et al, 1989;Campbell-Burk, 1989;Redfield and Papastavros, 1990;Vo et al, 2013). A resonance similar to peak A has previously been observed but not assigned.…”

Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.

“…In particular a change in protein conformation about the GDP p-phosphate is seen. NMR data also suggests small structural differences between wild-type p2lNRAS and the mutant [Aspl2Ip21NRAS in the GDP states (22,23). The crystal structure has now been obtained with p[NH]ppG bound (19), and this will be important in comparing different structural states due to different bound nucleotides.…”

“…Aliquots from these reaction mixtures were analyzed by HPLC to obtain Fig. 2 There is evidence (19)(20)(21)(22)(23) of conformation differences, either between GTP-and GDP-bound forms or between wild-type and mutant proteins. Conformational differences between p21NIAS.GTP and p21NRAS-GDP have been shown using circular dichroism measurements (20).…”

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

“…Although this is a very powerful method to identify selected amide resonances, it has the disadvantages that many labeled protein samples are required and that the lJCN coupling constant is comparable to the I5N line widths in the w1 dimension of HMQC spectra. This latter problem may be alleviated by several approaches, including using HSMQC experiments, direct I5N detection (Leighton & Lu, 1987), or I3C-I5N HMQC experiments (Westler et al, 1988), or by comparing spectra acquired with and without I3C decoupling (Campbell Burk et al, 1989).…”