Addition of isoprenoid lipids (prenylation) is critical from the activity of a number of enzymes that play essential roles in signal transduction pathways or membrane trafficking. Three protein prenyl transferases have been identified; a farnesyl transferase (FTase) which adds a 15-carbon isoprenoid, and two geranylgeranyl transferases (GTase-I, and -II) which add a 20-carbon isoprenoid. FTases and GTase-I modify a conserved cysteine residue located in a C-terminal tetrapeptide (""""""""CAAX"""""""" motif) of the modified protein. Prenylation of a number of small regulatory G proteins activate and target these to their cell membranes. Of particular medical relevance is the recent observation that Ras oncogen proteins are farnesylated. This modification is absolutely required for the transforming activity this protein. Treatment of Ras transformed cells with inhibitors to FTase has been shown to result in reversion of the transformed phenotype in tissue culture cells. Inhibition of Ras farnesylation is currently considered one of the most promising anti-cancer targets. Roughly 30% of human carcinomas are associated with oncogenic forms of Ras. Elucidation of the three dimensional structure of FTase is therefore important both for understanding fundamental processes in signal transduction and for the development of anti-cancer drugs or derivatives of existing drugs. The goal of this proposal is to understand the mechanism and substrate specificity of protein prenyl transferase proteins in terms of their three- dimensional structure.
The specific aims are summarized as follows: 1. To determine and refine the three-dimensional structures of farnesyl transferase together with appropriate substrate complexes by X-ray crystallography. Crystals of mammalian FTase and FTase with a bound peptide substrate have been grown that diffract to better than 2.5 A resolution and a native date set has been collected. To our knowledge this is the first prenyl transferase enzyme crystallized. The structure will provide a basis for understanding and interpreting biochemical and biophysical data on protein prenylation. 2. To determine the co-crystal structure of a ternary compllex of FTase, peptide substrate, and a farnesyl diphosphate analog. To determine the crystal structures of other complexes of FTase with appropriate substrates and inhibitors: peptides, farnesyl diphosphate, peptidomimetic and other inhibitors. These structures are essential for understanding the catalytic mechanism in atomic detail and determining the nature of substrate specificity. Together these structures provide a structural foundation for the design of improved anti-cancer therapeutics. 3. A long range goal is also to obtain crystals and determine the crystal structure of the GGTase-1 together with appropriate substrate complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052382-03
Application #
2415290
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1995-05-24
Project End
1999-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
3
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
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