SCIENCE

Structure of Mutant Human Oncogene Protein Determined

Primary structural differences between normal and transforming human c-Ha-tas oncogene proteins are localized in the loop regions (red) that interact with the β-phosphate ofguanosine diphosphate (tan)

The protein encoded by a mutant human oncogene differs only slight­ly in structure from the native protein that initiates normal cell di­vision, a finding that may compli­cate efforts to develop inhibitors of the mutant protein, according to chemists at the University of Cali­fornia, Berkeley.

Sung-Hou Kim, a Berkeley chem­istry professor and senior scientist at Lawrence Berkeley Laboratory, last year reported the x-ray struc­ture of the protein encoded by the normal c-Ha-ras gene, a protein be­lieved to signal cells to start or stop dividing through its interaction with guanosine triphosphate (GTP).

Kim has now determined the structure of the protein encoded by a transforming c-Ha-ras oncogene in which a valine codon replaces the normal glycine codon at posi­tion 12 in the gene [Nature, 337, page 90 (1989)]. The differences in the structures of the mutant and normal proteins are located primar­ily in a loop that interacts with the β-phosphate of a bound guanosine diphosphate (GDP) molecule.

Working with Kim at Berkeley were graduate students Liang Tong and Michael V. Milburn, postdoc­toral fellow Abraham M. de Vos, and laboratory chemist Jarmila Jancarik. Kim collaborated in the research with Susumu Nishimura and Shigeru Noguchi at the Na­tional Cancer Center Research In­stitute, Tokyo, and Eiko Ohtsuka and Kazunobu Miura at Hokkaido Uni­versity, Sapporo. The research was supported by the National Institutes of Health; Department of Energy; Japanese Ministry of Health & Welfare; and Merck, Sharp & Dohme.

In a healthy cell, the normal ras protein binds with GTP and trans­mits signals from the cell membrane to the cell interior that initiate a

cascade of reactions that cause the cell to divide, Kim explains. This ras protein exhibits GTPase activity, and in the normal course of events, the protein cleaves a phosphate ion from the bound GTP to produce GDP, which signals the cell to stop dividing.

The mutant ras protein, which has been found in almost all pancreas tumors and about half of colon tu­mors as well as tumors from the breast, bladder, and lung, appears to have a significantly reduced GTPase activity. Kim says that the rate of conversion of GTP to GDP in the mutant protein is less than 5% of what it is in the normal protein.

The recently reported research suggests why this might be so. The ras protein contains six β-strands, four α-helices, and nine connecting loops. The protein consists of two structural domains—the amino-

terminal domain, containing the first 75 amino acid residues (including the first three β-strands and one α-helix), which binds phosphate; and the carboxy-terminal domain (including the last three β-strands and three α-helices), which recog­nizes guanine.

The largest differences in struc­ture were found in the first loop in the amino-terminal half of the mol­ecule, corresponding to amino acid residues nine to 18, Kim says. Tak­en together, these differences re­sult in a loop that is about 2 A larger in the mutant protein than the corresponding loop in the nor­mal protein. This results in the loss of two hydrogen bonds from the protein backbone amino groups of residues 12 and 13 to the /3-phos-phate of the bound GDP molecule. (The x-ray structure was determined for the protein with a bound GDP.)

Kim had suggested previously that

January 16, 1989 C&EN 31

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Science

this loop would straddle the phos-phodiester bond between the β- and γ-phosphates of GTP. This bond is the prime candidate to be the cata­lytic site for GTP hydrolysis in the normal protein. The loss of two hy­drogen bonds may alter the orien­tation of the jS-phosphate group, Kim says, and thus somehow inter­fere with the hydrolytic reaction.

Working out the complete mech­anism of loss of GTPase activity will require determination of the struc­ture of native and mutant ras pro­teins bound to GTP. This effort is complicated by the proteins' hydrol­ysis of GTP. Thus the work must be carried out on crystals of the pro­teins bound to GTP analogs that

contain a bond between the β- and 7-phosphates that is not susceptible to hydrolysis. Efforts to produce crystals of such complexes are un­der way in Kim's laboratory.

The similarity of structures be­tween the mutant and the normal ras proteins may complicate efforts to develop a drug to deactivate the mutant protein and thus halt tumor growth. "The drug must deactivate the mutant form of the protein with­out deactivating the normal protein, which is essential to normal cell growth," Kim points out. "It's going to be a difficult job, but it's theoret­ically possible. Chemists are pretty clever."

Rudy Baum

EDUCATION

Management-of-technology program debuts Satellite broadcasting signals this month are carrying the first courses in a new master of science program initiated by National Technological University in management of tech­nology. NTU plans to have the com­plete two-year program available in June.

NTU, based in Fort Collins, Colo., is the first electronic university ded­icated to nationwide satellite broad­casting of graduate-level advanced engineering and technical educa­tion. An accredited, private, non­profit institution founded in 1984, it is a university without a campus or permanent faculty, and is de­signed to serve the advanced edu­cation needs of busy, mobile engi­neers, scientists, and technical man­agers. Ranging from the University of Massachusetts in the Northeast to the University of Arizona in the Southwest, some 28 institutions par­ticipate in the network.

So far, NTU's focus has been fair­ly electrical in nature. The new pro­gram brings the number of M.S. programs offered to six. The other five are computer engineering, com­puter science, electrical engineer­ing, engineering management, and manufacturing systems engineering.

"Management of technology has been identified as one of the top national priorities, especially in view

of the intensity of international com­petition," says Robert W. DeSio, vice president for development and long-range planning for NTU. "Because management of technology is a rel­atively new discipline and interdis­ciplinary in nature, a traditional uni­versity would have difficulty in bringing together the required ed­ucational programs," he adds.

NTU is in the second phase of development of the new program. The first phase focused on format and curriculum.

Students will be nominated to the program by each of NTU's 60 spon­soring corporations and government agencies. They will be managers who are expected to advance into senior corporate management dur­ing their careers. They will have the financial support of their cor­porations or agencies and will be freed from enough of their regular responsibilities to finish the pro­gram in 24 months.

Students will as usual take courses at their work sites through NTU's instructional television network. But in a departure from the format of other NTU programs, they will be brought together during the two-year period for seven one-week in­tense residencies at different par­ticipating university campuses.

James Krieger