Crystal Structure Of Protein Farnesyltransferase At 2.25 Å Resolution. Hee-Won Park1, Stephen B. Long1, Sobha R. Boduluri1, John F. Moomaw2, Patrick J. Casey2, and Lorena S. Beese1 1Dept. of Biochem., Duke Univ. Medical Center 2Dept. of Molecular Cancer Biology, Duke Univ. Medical Center.
Protein farnesyltransferase (FTase) catalyzes the carboxy-terminal lipidation of Ras and several other cellular signal transduction proteins. The essential nature of this modification for proper function of these proteins has led to the emergence of FTase as a target for the development of new anti-cancer therapy. Inhibition of this enzyme suppresses the transformed phenotype in cultured cells and causes tumor regression in animal models.
Ftase is a heterodimer consisting of 48kD (a) and 46kD ([beta]) subunits; the same alpha subunit is also a component of a related enzyme, protein geranylgeranyltransferase type I (GGTase-I). Both FTase and GGTase-I use an isoprenoid diphosphate (FPP or GGPP respectively) to modify the substrate via a thioether linkage to an invariant cysteine residue fourth from the carboxy-terminus in a sequence motif commonly referred to as the "CaaX box". In this motif the second and third residues are small aliphatic amino acids. The two zinc metalloenzymes are specific for the C-terminal residue. FTase recognizes proteins that contain Ser, Met, Ala, or Gln at this position, while GGTase-I prefers proteins that terminate in Leu. A number of studies support a direct involvement of the zinc ion in catalysis; the most compelling being the recent finding that the thiol group of a CaaX peptide substrate directly coordinates the metal atom in a ternary complex.
We have determined the structure of rat FTase, which shares 97% sequence identity with the human enzyme. FTase crystallizes in space group P65 with the unit cell dimensions a=b= 167.06 Å, and c= 97.90 Å. Its crystal structure was determined at 2.25 Å resolution by multiple isomorphous replacement and anomalous scattering with three heavy atom derivatives, solvent flattening, phase combination, and crystallographic refinement. The refined atomic model has an R-value of 21.7%.
The crystal structure shows a combination of two unusual domains: a crescent-shaped seven helical hairpin domain and an a-a barrel domain. The active site is formed by two clefts that intersect at a bound zinc ion. One cleft contains a nine-residue peptide that may mimic the binding of the Ras substrate; the other cleft is lined with highly conserved aromatic residues appropriate for binding the farnesyl isoprenoid with required specificity.