May 23, 1983 - ... Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Building ..... DeFeo, D., Gonda, M. A., Young, H. A., Chang, E. H., Lowy,.
Proc. Nati. Acad. Sci. USA Vol. 80, pp. 5253-5257, September 1983
Biochemistry
Prediction of the three-dimensional structure of the transforming region of the EJ/T24 human bladder oncogene product and its normal cellular homologue (conformational energy/protein p21/transformation)
MATTHEW R. PINCUSt, JOS VAN RENSWOUDEt, JOE B. HARFORDt, ESTHER H. CHANG§, ROBERT P. AND RICHARD D. KLAUSNERt
CARTYT,
*Laboratory of Biochemistry and Metabolism, National Institutes of Health, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, Building 10, Room 9N-119, Bethesda, Maryland 20205; tDepartment of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10023; §Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814; and ¶Department of Biochemistry,
State University of New York, Downstate Medical Center, Brooklyn, New York 11203
Communicated by J. E. Rall, May 23, 1983
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. 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 21,000dalton 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 (12-14). The normal cellular counterpart of a tumor oncogene is termed a proto-oncogene. One of the cloned human c-onc (c-Ha ras1) genes appears to be identical to the proto-oncogene of the bladder carcinoma oncogene (9, 12-14). The EJ/T24 bladder carcinoma oncogene and its proto-oncogene analogue are remarkably similar in structure as judged The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
by restriction mapping (12-14). 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 (1517), 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 1
10
Met-Thr-Glu-Tyr-Lys-Leu-Val-Val-Val-Gly-Ala-Gly20
Gly-Val-Gly-Lys-Ser-Ala-Leu-Thr. The hydrophobic sequence for which we calculate the structure extends from residue 6 through residue 15. Other point mutations that drastically affect function occur in a number of proteins, the most familiar of which is the substitution of valine for glutamate at position 6 in sickle cell hemoglobin. This substitution is thought to result in an altered interaction of hemoglobin monomers rather than in major changes in the conformation of the monomer itself. Thus, it would be desirable to determine whether the point mutation in the tumor p21 can result in structural changes in the oncogene-encoded protein. Such a change may be related to the process of transformation. METHODS Using the potential energy function of the empirical conformational energy for polypeptides and proteins (ECEPP) program (18), computer-based techniques for the construction of the most probable three-dimensional structures of long polypeptides from their amino acid sequences have been developed (18-20). These techniques have been applied to hydrophobic peptides (up to 26 amino acids) and yield structures that agree very well with available experimental data (18-20). Because of the small partial atomic charges, which greatly limit their solAbbreviations: Ha-MuSV, Harvey murine sarcoma virus; p21, the 21,000dalton protein.
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Biochemistry: Pincus et al.
we are now calculating only the conformation of a hydrophobic region that we assume to be shielded from solvent as for example in the hydrophobic core of a lipid bilayer. Three basic steps are involved in these structure predictions. First, we identify probable nucleation sequences for folding. Usually such sequences consist of several consecutive hydrophobic residues. The preferred conformations of the diand tripeptide subsequences of these nucleation segments are then calculated. This step is accomplished by combining all of the single-residue conformational energy minima (21) of the individual amino acids and subjecting these combinations to an energy-minimization protocol in which all of the dihedral angles of the peptide are allowed to vary. All structures whose calculated conformational energies are within a selected energy difference from that of the global minimum (i.e., the lowest energy conformation) are retained. Any structure whose calculated energy is within 3 kcal/mol (1 cal = 4.18 J) of the global minimum is taken as an "allowed" conformation and is used as a starting conformation for the subsequent chain build-up (19, 20). The resulting allowed low-energy conformations for the diand tripeptides are then combined using the method of combination of nondegenerate minima (19, 20), in which only those structures with different backbone conformations are included. These combinations are then energy minimized and the above process is repeated. In this way, succeeding di- and tripeptides are added to the calculated structure of the growing polypeptide chain. Once this computation has been completed for the nucleation sequences that themselves have, at most, a few allowed conformations, the intervening and flanking sequences are added as di- or tripeptides to the ends of the nucleation sequences. At all stages, all of the dihedral angles of the chain are allowed to vary in the energy-minimization protocol, and all interatomic energies are explicitly evaluated. Thus, short-, medium-, and long-range interactions are included throughout the process. At each stage of the chain build-up process, the amino and carboxyl termini contained N-acetyl and NHCH3 groups, respectively, to include end effects of neighboring residues not included in a particular peptide.
vent interactions,
Proc. Natl. Acad. Sci. USA 80 (1983)
RESULTS We have computed the allowed conformations of the hydrophobic decapeptide comprised of residues 6-15 of both normal and tumor p21. These sequences were predicted from nucleic
acid sequence analysis of the bladder carcinoma oncogene and its homologue in normal cells (15-17). In our calculation, the tetrapeptide Leu-(Val)3 (residues 6-9) was selected as a nucleation sequence. The allowed conformations of this peptide were then combined with the less structured (residues 10 and 11). Because the alteration in the tumor p21 sequence is located at residue 12 (15-17) either glycine or valine was added to all of the allowed conformations of the hexapeptide (residues 611). The remaining three amino acids-glycine, valine, glycine (residues 13-15)-were added successively as single amino acids to each of the nondegenerate conformers of the preceding peptide. In calculating the allowed conformations of the nucleation tetrapeptide, we combined the nondegenerate low-energy conformations of Leu-Val and Val-Val and subjected this combination to energy minimization. The global minimum is an a-helix, although other forms exist with a reasonable energy difference of the global energy minimum. Most of these other low-energy conformers are a-helical for at least the first three residues. Interestingly, on replacement of valine by isoleucine, an apparently conservative substitution, the calculation yields an almost fully extended chain for Leu-(Ile)3 rather than the a-helix of Leu-(Val)3. Thus, the structure of the hydrophobic tetrapeptide (residues 6-9) is highly sequence specific. It is clear that Leu(Val)3 could provide an a-helical nucleating sequence for folding of the remaining less-structured peptide Gly-Ala-Gly(Val)-GlyVal-Gly. When we combined the minima for Leu-(Val)3 with those of Gly-Ala, we obtained nine nondegenerate low-energy minima for the resulting hexapaptide. Again, the global minimum and many of the low-energy forms were a-helical. However, in contrast to Leu-(Val)3, non-a-helical forms do appear. Addition of glycine or valine to this hexapeptide yields very different sets of heptapeptide structures after energy minimization. For the
Table 1. Low-energy conformations for the hydrophobic decapeptide in the "normal" protein N-acetyl-Leu-(Val)3-Gly-Ala-Gly-Gly-Val-Gly-NHCH3 Energy, Conformational state Conformert Leu Val Val Val Gly Ala Gly-12 Gly Val Gly kcal/mol A 0.0 A C D* A A A C A A 1 A A 0.8 D* A A A C A A A 2 F* A 0.9 C D D* A D* A A E 3 A* 1.2 A D D* D A A A A A 4 A* A 1.2 D* A A C A A A A 5 A* 1.6 D* A A A A C A A A 6 D* 1.7 A D D* C A E D* A A 7 D* 1.7 D* A A A A A C A A 8 A A A 1.9 D* A A A C A A 9 1.9 D A D* C A A A A A A 10 2.0 E* C A D* D 11 A A A E D* 2.0 A A A A A A A A 12 A A Low-energy conformations are defined as those whose energies lie within 2 kcal/mol ofthat of the global minimum (conformer 1). Conformational states are defined as follows: States in which the dihedral angles are represented by a single-letter code are defined in refs. 18 and 22. The familiar states are A (a-helix) and E (extended). States F, E*, F, F*, and D and D* are collectively called "beta" conformations. The actual dihedral angle ranges for all single-letter states are A, -110°
