Structural effects of substitutions on the p21 proteins.

Abstract

The conformational effects of different amino acid substitutions (lysine, serine, proline, and D-valine) for glycine at position 12 in the p21 oncogene-encoded proteins have been investigated by using conformational energy calculations. The normal cellular gene codes for a glycine at position 12 in the amino acid sequence, in the middle of a hydrophobic p21-(6-15)-decapepetide from Leu-6 to Gly-15. Mutations that cause amino acid substitutions for Gly-12 result in a protein product that produces malignant transformation of cells. We now find that not only are the preferred structures for the lysine-and serine-containing peptides more restricted and more helical than those for the glycine-containing peptide, but the lowest-energy structure for each substituted peptide is exactly the same as that previously found for the peptide with Val-12, suggesting the existence of a "malignancy-causing" conformation. None of the preferred conformations for the valine-, lysine-, and serine-containing peptides contain chain reversals at positions 11 and 12. However, we find that proline, unlike these residues but like glycine, at position 12 causes helix termination at positions 11 and 12, a result that suggests that the p21 protein product with proline at position 12 may exhibit lowered transforming potential, in agreement with the results of recent genetic recombination experiments.Human oncogenes in the ras family are homologous to a normal cellular protooncogene that codes for a normal protein product of Mr 21,000 (p21 protein), and several of them such as the EJ bladder carcinoma oncogene (1, 2) differ from it in only the 12th coding triplet, which in the protooncogene is GGC, the codon for glycine (1,2). Amino acid substitutions at position 12 in the p21 protein encoded by human ras oncogenes are known to result in abnormal cellular control and ultimately to cause malignant transformation of cells (1-3).The sequence of the first 20 amino acids (1, 2) of the p21 protein encoded by the protooncogene is:Val-Gly-Lys-Ser-Ala-Leu-Thr.Malignant transformation of cells occurs when the glycine at position 12 is replaced by a variety of amino acids (1, 2, 4, 5).This Gly-12 occurs in the middle of a long sequence (the transforming region) of 10 hydrophobic or nonpolar amino acid residues, the only such sequence in the p21 proteins.We have shown previously that glycine at position 12 shows a marked preference for the D* (conformations are explained in the legend to Table 1) conformation that is inaccessible to all naturally occurring L-amino acids (6-8). This result suggested to us that substitution of any other amino acid for glycine at position 12 would result in a mandatory change in chain conformation in the region of residue 12 that may result in malignant transformation (6-8).We also found that, for the "normal" p21 protein, a slightly higher energy conformation existed that was identical to the lowest energy conformer for the transforming p21 protein with valine at position 12 in which the chain reversal (CD*) conform...

Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site.

“…They are believed to play an essential role in growth and/or development. A number of structural comparisons and structure predictions have been published in an attempt to explain the structural implications of the amino acid replacements at positions 12, 13 and 59,61, which lead to tumorigenic activation of the protein (Gay and Walker, 1983;Wierenga and Hol, 1983; Leberman and Egner, 1984;Halliday, 1983;Pincus and Brandt-Rauf, 1985;Murakami, 1985). Since secondary structure predictions at the very best are only 60% accurate and are sometimes contradictory to each other, the actual determination of the three-dimensional structure is essential.…”

Conformational effects of substituting amino acids for glutamine-61 on the central transforming region of the P21 proteins.

“…In a series of studies (13)(14)(15)(16) in which the preferred conformations of the amino-terminal peptide were explored using conformational energy calculations (16), it was found that substitution of any L-amino acid, except Pro, for Gly-12 (which occupies this position in the normal protein) caused a well-defined change in the structure of the peptide, irrespective of the substituting amino acid. This change was such that a chain-reversal (bend) conformation, that occurred at Ala-11-Gly-12 in the normal protein, now occurred at Xaa-12-Gly-13, where Xaa is the amino acid substituting for Gly-12.…”

“…This change was such that a chain-reversal (bend) conformation, that occurred at Ala-11-Gly-12 in the normal protein, now occurred at Xaa-12-Gly-13, where Xaa is the amino acid substituting for Gly-12. The peptides with arbitrary amino acid substitutions at position 12 could not adopt the normal lowest energy Ala-11-Gly-12 bend, whereas the "normal" peptide could adopt the lowestenergy structure for the substituted peptides, i.e., a bend at positions 12 and 13, at a sacrifice of several kcal (1 cal = 4.184 J) in conformational energy (14,15). On the basis of these results, it was predicted that P21 proteins containing any L-amino acid, except Pro, at position 12, would cause malignant transformation of cells (14,15) and that high intracellular concentrations of the normal protein would likewise cause cell transformation since significant levels of the alternate "malignancy-causing" conformation (bend at positions 12 and 13) would then be expressed (14).…”

“…The peptides with arbitrary amino acid substitutions at position 12 could not adopt the normal lowest energy Ala-11-Gly-12 bend, whereas the "normal" peptide could adopt the lowestenergy structure for the substituted peptides, i.e., a bend at positions 12 and 13, at a sacrifice of several kcal (1 cal = 4.184 J) in conformational energy (14,15). On the basis of these results, it was predicted that P21 proteins containing any L-amino acid, except Pro, at position 12, would cause malignant transformation of cells (14,15) and that high intracellular concentrations of the normal protein would likewise cause cell transformation since significant levels of the alternate "malignancy-causing" conformation (bend at positions 12 and 13) would then be expressed (14). The former conclusion was corroborated in site-specific mutagenesis experiments in which it was shown that cells transfected with a series ofras genes coding for proteins with various naturally occurring amino acid residues at position 12 underwent malignant transformation with all but the genes coding for Gly and Pro (5).…”

“…The central transforming region contains mostly polar residues, unlike the amino-terminal transforming region that contains a long (10-residue) sequence of nonpolar amino acids from Leu-6 to Gly-15, which is highly structured (14,15,18). The sequence from Ile-55 to Arg-68 is Ile-Leu-Asp-ThrAla-Gly-Gln-Glu-Glu-Tyr-Ser-Ala-Met-Arg.…”