Prediction of the three-dimensional structure of the transforming region of the EJ/T24 human bladder oncogene product and its normal cellular homologue.

Abstract

The three-dimensional structures of the transforming region of the product of the EJ/T24 human bladder oncogene and of the c-Ha ras8-gene product have been calculated by using conformational energy calculations. These two genes, representing a transforming oncogene and its normal cellular homologue, encode 21,000-dalton peptides that differ by one amino acid at position 12. We therefore examined the energetically allowed conformations of the hydrophobic decapeptide surrounding this substitution site. The calculations show that the most favorable form of the c-Ha ras-l gene product exists when glycine-12 is in a left-handed bend conformation. No other amino acid can adopt this conformation and thus the bladder oncogene peptide containing valine at position 12 has a markedly different three-dimensional structure. A simple model is proposed to account for the consequences of a position 12 mutation.by restriction mapping (12)(13)(14). In our routine DNA transfection assays, however, the cloned oncogene has a transforming potency that is several orders of magnitude higher than that of cloned c-Ha ras-1. By chimeric reconstruction, this difference in transforming potency was localized to a 350-nucleotide sequence in the 5' end of the p21 coding sequences. The sequences of these regions of DNA have been determined (15)(16)(17), and the difference in transforming potential was pinpointed to a single-base change-from guanosine to thymidine. This mutation results in substitution of valine for glycine at residue 12 of the predicted amino acid sequence of p21 and occurs in the middle of a hydrophobic sequence. The sequence for the first 20 amino acid residues for the normal (nontransforming) protein is Harvey murine sarcoma virus (Ha-MuSV) was originally isolated from a tumor-bearing rat that had been inoculated with Moloney murine leukemia virus (1). The resultant retrovirus induces solid tumors and erythroleukemia in susceptible mice (2), and the naked viral DNA is capable of transforming NIH 3T3 cells after transfection (3). The region of the viral DNA responsible for the transforming activity has been localized to a one-kilobase segment of nucleic acid derived from the original host rat. A 21,000-dalton protein (p21) is encoded by this region of the viral DNA (4-7). Sequences homologous to the Ha-MuSV transforming region have been molecularly cloned from normal rat DNA (8). Moreover, homology between these sequences and DNA from other species has allowed the identification and molecular cloning from normal human DNA of genes related to the oncogene of Ha-MuSV (9). These cellular homologues (conc or more specifically in this case c-Ha ras) encode a 21,000-dalton protein immunologically and biochemically related to viral p21 (10).Through DNA transfer techniques, oncogenic sequences have been identified in several human tumors (11). The tumor oncogenes found in T24 and EJ human bladder carcinoma cell lines have been shown to be related to the c-Ha ras gene (12)(13)(14). The normal cellular counterpart of a tumor o...

Structural effects of substitutions on the p21 proteins.

“…These results further suggested that if the "normal" p21 protein were to be present at elevated intracellular levels, where the alternate (higher energy) conformation would exist in significant concentration, malignant transformation likewise would take place. The results of experiments in which malignant transformation of NIH 3T3 cells transfected with the protooncogene spliced to a viral long terminal repeat occurred (3) are fully compatible with these computed results (6)(7)(8).In this paper we examine the structural effects of substituting other amino acids besides valine at position 12, specifically lysine, serine, proline, and the D-form of valine. We compare our results with those obtained for proteins containing glycine and valine at this position (6-8) and with experimental results on the ability of each protein with a different amino acid at position 12 (1, 2, 4, 5) to transform cells.…”

“…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)(7)(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*) conformation occurred at residues Val-Gly at positions 12 and 13.…”

“…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)(7)(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)(7)(8).…”

Phosphate‐binding sequences in nucleotide‐binding proteins

“…Earlier model calculations of the threedimensional structure of the PEJ/T24 oncogene product indicate that the Gly 12 peptide has a conformation and flexibility which are larger than those of the Val 12 peptide [12]. These differences may well influence the rate of transition between a GTP and GDP conformation of the protein.…”

Export of protein in Escherichia coli: a novel mutation in ompC affects expression of other major outer membrane proteins

“…Third, Gly is the only naturally occurring amino acid which lacks a chiral carbon, and this allows it to assume a unique conformational state (the D* or left-handed 3 conformation) in the polypeptide backbone. Mutational replacement of glycine has recently been shown to have a strong effect on the minimum energy conformation of a polypeptide backbone which paralleled a change in biological function (26).…”