Comparison of the low energy conformations of an oncogenic and a non-oncogenic p21 protein, neither of which binds GTP or GDP

Oncogenic p21 protein, encoded by theras-oncogene, that causes malignant transformation of normal cells and many human tumors, is almost identical in sequence to its normal protooncogene-encoded counterpart protein, except for the substitution of arbitrary amino acids for the normally occurring amino acids at critical positions such as Gly 12 and Gin 61. Since p21 is normally activated by the binding of GTP in place of GDP, it has been postulated that oncogenic forms must retain bound GTP for prolonged time periods. However, two multiply substituted p21 proteins have been cloned, neither of which binds GDP or GTP. One of these mutant proteins with Val for Gly 10, Arg for Gly 12, and Thr for Ala 59 causes cell transformation, while the other, similar protein with Gly 10, Arg 12, Val for Gly 13 and Thr 59 does not transform cells. To define the critical conformational changes that occur in the p21 protein that cause it to become oncogenic, we have calculated the low energy conformations of the two multiply substituted mutant p21 proteins using a new adaptation of the electrostatically driven Monte Carlo (EDMC) technique, based on the program ECEPP. We have used this method to explore the conformational space available to both proteins and to compute the average structures for both using statistical mechanical averaging. Comparison of the average structures allows us to detect the major differences in conformation between the two proteins. Starting structures for each protein were calculated using the recently deposited x-ray crystal coordinates for the p21 protein, that was energy-refined using ECEPP, and then perturbed using the EDMC method to compute its average structure. The specific amino acid substitutions for both proteins were then generated into the lowest energy structure generated by this procedure, subjected to energy minimization and then to full EDMC perturbations. We find that both mutant proteins exhibit major differences in conformation in specific regions, viz., residues 35–47, 55–78, 81–93, 96–110, 115–126, and 123–134, compared with the EDMC-refined x-ray structure of the wild-type protein. These regions have been found to be the most flexible in the p21 protein bound to GDP from prior molecular dynamics calculations (Dykeset al., 1993). Comparison of the EDMC-average structure of the transforming mutant with that of the nontransforming mutant reveals major structural differences at residues 10–16, 32–40, and 60–68. These structural differences appear to be the ones that are critical in activation of the p21 protein. Analysis of the correlated motions of the different regions of the two mutant proteins reveals that changes in the conformation of regions in the carboxyl half of the protein are caused by changes in conformation around residues 10–16 and are transmitted by means of residues around Gln 61. The latter region therefore constitutes a “molecular switch” unit, in agreement with conclusions from prior work.

[1]  M. Pincus,et al.  Prediction of the three-dimensional structure of the transforming region of the EJ/T24 human bladder oncogene product and its normal cellular homologue. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[2]  N Go,et al.  Structural basis of hierarchical multiple substates of a protein. I: Introduction , 1989, Proteins.

[3]  H. Scheraga,et al.  Monte Carlo-minimization approach to the multiple-minima problem in protein folding. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Elledge,et al.  Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1 , 1993, Nature.

[5]  D. Blair,et al.  Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis , 1987, Molecular and cellular biology.

[6]  D. Lowy,et al.  Guanosine triphosphatase activating protein (GAP) interacts with the p21 ras effector binding domain. , 1988, Science.

[7]  M. Pincus,et al.  Conformational effects of substituting amino acids for glutamine-61 on the central transforming region of the P21 proteins. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[8]  W. Kabsch,et al.  Refined crystal structure of the triphosphate conformation of H‐ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. , 1990, The EMBO journal.

[9]  S. Halegoua,et al.  Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21 , 1986, Nature.

[10]  M. Pincus,et al.  Inhibition of ras-oncogene-encoded p21 protein-induced maturation of oocytes by p21 peptide sequences predicted to be effector domain sites by molecular modelling , 1992 .

[11]  M. Weber,et al.  Complexes of Ras.GTP with Raf-1 and mitogen-activated protein kinase kinase. , 1993, Science.

[12]  F. McCormick,et al.  A cytoplasmic protein stimulates normal N-ras p21 GTPase, but does not affect oncogenic mutants. , 1987, Science.

[13]  H. Scheraga,et al.  Empirical solvation models can be used to differentiate native from near‐native conformations of bovine pancreatic trypsin inhibitor , 1991, Proteins.

[14]  Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure , 1990, Journal of protein chemistry.

[15]  S H Kim,et al.  Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP. , 1991, Journal of molecular biology.

[16]  M. Wigler,et al.  RAS proteins can induce meiosis in xenopus oocytes , 1985, Cell.

[17]  M. Pincus,et al.  Molecular dynamics of the H-ras gene-encoded p21 protein; identification of flexible regions and possible effector domains. , 1993, Journal of biomolecular structure & dynamics.

[18]  M. Pincus,et al.  The structure of the amino terminal transforming segment of the p21 protein, Tyr4-Thr20 (with Asp12), by two-dimensional NMR. , 1988, Biochemical and biophysical research communications.

[19]  H. Scheraga,et al.  Energy parameters in polypeptides. 10. Improved geometrical parameters and nonbonded interactions for use in the ECEPP/3 algorithm, with application to proline-containing peptides , 1994 .

[20]  D. Shibata,et al.  Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes , 1988, Cell.

[21]  C. Marshall,et al.  All ras proteins are polyisoprenylated but only some are palmitoylated , 1989, Cell.

[22]  J. L. Bos,et al.  Two dominant inhibitory mutants of p21ras interfere with insulin-induced gene expression , 1991, Molecular and cellular biology.

[23]  M. Pincus,et al.  A peptide from the GAP-binding domain of the ras-p21 protein and azatyrosine block ras-induced maturation of Xenopus oocytes. , 1991, Anticancer research.

[24]  H. Scheraga,et al.  Energy parameters in polypeptides. 9. Updating of geometrical parameters, nonbonded interactions, and hydrogen bond interactions for the naturally occurring amino acids , 1983 .

[25]  H A Scheraga,et al.  On the multiple‐minima problem in the conformational analysis of polypeptides. I. Backbone degrees of freedom for a perturbed α‐helix , 1987 .

[26]  H. Scheraga,et al.  On the multiple‐minima problem in the conformational analysis of polypeptides. II. An electrostatically driven Monte Carlo method—tests on poly(L‐alanine) , 1988, Biopolymers.

[27]  H A Scheraga,et al.  Empirical solvation models in the context of conformational energy searches: Application to bovine pancreatic trypsin inhibitor , 1992, Proteins.

[28]  M Geyer,et al.  Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras. , 1994, Biochemistry.

[29]  D. Lowy,et al.  The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH , 1991, Molecular and cellular biology.

[30]  W. Kabsch,et al.  Three-dimensional structures of H-ras p21 mutants: Molecular basis for their inability to function as signal switch molecules , 1990, Cell.

[31]  M. Pincus,et al.  Inhibition of ras oncogene-encoded P21 protein-induced pinocytotic activity by a synthetic peptide corresponding to an effector domain of the protein , 1990 .

[32]  D. Burstein,et al.  Activated N‐ras gene induces neuronal differentiation of PC12 rat pheochromocytoma cells , 1986, Journal of cellular physiology.

[33]  William E. Grizzle,et al.  Detection of high incidence of K-ras oncogenes during human colon tumorigenesis , 1987, Nature.

[34]  S. Rackovsky,et al.  Prediction of protein conformation on the basis of a search for compact structures: Test on avian pancreatic polypeptide , 1993, Protein science : a publication of the Protein Society.

[35]  M. Wigler,et al.  Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. , 1993, Science.

[36]  S. Rackovsky,et al.  Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein. , 2009, International journal of peptide and protein research.

[37]  D. Lowy,et al.  Identification of small clusters of divergent amino acids that mediate the opposing effects of ras and Krev-1. , 1990, Science.

[38]  D. Osguthorpe,et al.  Structure and energetics of ligand binding to proteins: Escherichia coli dihydrofolate reductase‐trimethoprim, a drug‐receptor system , 1988, Proteins.

[39]  Harold A. Scheraga,et al.  Standard‐geometry chains fitted to X‐ray derived structures: Validation of the rigid‐geometry approximation. I. Chain closure through a limited search of “loop” conformations , 1991 .

[40]  H. Kung,et al.  Insulin induction of Xenopus laevis oocyte maturation is inhibited by monoclonal antibody against p21 ras proteins. , 1987, Molecular and cellular biology.