The crystal structure of human protein farnesyltransferase reveals the basis for inhibition by CaaX tetrapeptides and their mimetics

Protein farnesyltransferase (FTase) catalyzes the attachment of a farnesyl lipid group to the cysteine residue located in the C-terminal tetrapeptide of many essential signal transduction proteins, including members of the Ra s superfamily. Farnesylation is essential both for normal functioning of th ese proteins, and for the transforming activity of oncogenic mutants. Conse quently FTase is an important target for anti-cancer therapeutics. Several FTase inhibitors are currently undergoing clinical trials for cancer treatm ent. Here, we present the crystal structure of human FTase, as well as tern ary complexes with the TKCVFM hexapeptide substrate, CVFM non-substrate tet rapeptide, and L-739,750 peptidomimetic with either farnesyl diphosphate (F PP), or a nonreactive analogue. These structures reveal the structural mech anism of FTase inhibition. Some CaaX tetrapeptide inhibitors are not farnes ylated, and are more effective inhibitors than farnesylated CaaX tetrapepti des. CVFM and L-739,750 are not farnesylated, because these inhibitors bind in a conformation that is distinct from the TKCVFM hexapeptide substrate. This non-substrate binding mode is stabilized by an ion pair between the pe ptide N terminus and the alpha-phosphate of the FPP substrate. Conformation al mapping calculations reveal the basis for the sequence specificity in th e third position of the CaaX motif that determines whether a tetrapeptide i s a substrate or non-substrate. The presence of beta-branched amino acids i n this position prevents formation of the non-substrate conformation; all o ther aliphatic amino acids in this position are predicted to form the non-s ubstrate conformation, provided their N terminus is available to bind to th e FPP alpha-phosphate. These results may facilitate further development of FTase inhibitors.