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Conformation of a heptapeptide substrate bound to protein farnesyltransferase
S J Stradley1, J Rizo, L M Gierasch
1Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041.
Abstract:
Protein farnesyltransferase catalyzes isoprenylation of the cysteine four residues from the C-terminus of several proteins including p21ras. Farnesylation is required for the transforming activity of Ras, and many efforts are underway to develop inhibitors of farnesyltransferase. We have used nuclear magnetic resonance spectroscopy to determine the farnesyltransferase-bound conformation of a heptapeptide substrate, KTKCVFM, which competes for the modification of p21Ha-ras in an in vitro assay. Analysis of transferred nuclear Overhauser effects reveals that the CVFM sequence of the peptide substrate is directly involved in binding to the enzyme and adopts a type I beta-turn conformation in the bound state. The present structural information should aid in the design of more effective inhibitors of the enzyme and in understanding the nature of the peptide binding site.
Insights
Nuclear magnetic resonance revealed the bound conformation of a peptide substrate for protein farnesyltransferase. This structural insight into farnesyltransferase inhibitors is crucial for cancer drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Enzyme Kinetics
Background:
- Protein farnesyltransferase (PFT) mediates the isoprenylation of cysteine residues in proteins like p21Ras.
- This farnesylation is essential for the oncogenic transforming activity of Ras proteins.
- Developing PFT inhibitors is a key strategy in cancer therapy research.
Purpose of the Study:
- To determine the enzyme-bound conformation of a heptapeptide substrate (KTKCVFM) of PFT.
- To elucidate the structural basis of peptide recognition by PFT.
- To provide insights for the rational design of PFT inhibitors.
Main Methods:
- Nuclear magnetic resonance (NMR) spectroscopy, specifically transferred nuclear Overhauser effects (trNOE).
- In vitro enzymatic assay using a peptide substrate that competes with p21Ha-ras modification.
- Structural analysis of the peptide-enzyme complex.
Main Results:
- The CVFM sequence of the KTKCVFM peptide is critical for binding to PFT.
- The bound peptide adopts a type I beta-turn conformation within the enzyme's active site.
- NMR data provided detailed structural information on the peptide-PFT interaction.
Conclusions:
- The determined structure of the bound peptide substrate offers a template for designing more potent and specific PFT inhibitors.
- Understanding the peptide binding site's conformation is vital for advancing PFT-targeted cancer therapies.
- This study enhances the understanding of enzyme-substrate interactions in protein isoprenylation.