Peptide Specificity of Protein Prenyltransferases Is Determined Mainly by Reactivity Rather than Binding Affinity

Abstract

Protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase I) catalyze the attachment of lipid groups from farnesyl diphosphate and geranylgeranyl diphosphate, respectively, to a cysteine near the C-terminus of protein substrates. FTase and GGTase I modify several important signaling and regulatory proteins with C-terminal CaaX sequences ("C" refers to the cysteine residue that becomes prenylated, "a" refers to any aliphatic amino acid, and "X" refers to any amino acid). In the CaaX paradigm, the C-terminal X-residue of the protein/peptide confers specificity for FTase or GGTase I. However, some proteins, such as K-Ras, RhoB, and TC21, are substrates for both FTase and GGTase I. Here we demonstrate that the C-terminal amino acid affects the binding affinity of K-Ras4B-derived hexapeptides (TKCVIX) to FTase and GGTase I modestly. In contrast, reactivity, as indicated by transient and steady-state kinetics, varies significantly and correlates with hydrophobicity, volume, and structure of the C-terminal amino acid. The reactivity of FTase decreases as the hydrophobicity of the C-terminal amino acid increases whereas the reactivity of GGTase I increases with the hydrophobicity of the X-group. Therefore, the hydrophobicity, as well as the structure of the X-group, determines whether peptides are specific for farnesylation, geranylgeranylation, or dual prenylation.

Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

“…This general observation is consistent with previous reports that rPFTase can prenylate peptide sequences not present in the genome, and most of these have been demonstrated to be substrates using the in vitro fluorescence assay. 32,33 Some of these sequences are found in the genomes of other organisms including Schizosaccharomyces pombe (CVIA), Schizophyllum commune (CVVA), Drosophila menanogaster (CVSS), Arabidopsis thaliana (CVTA), and Mus musculus (CVAS), whose prenylated proteins have not been studied in detail. Of potential greater significance are sequences found in human pathogens including CVFQ ( Shigella dysenteriae ) and CVTH ( Bacillus circulans ) as well as various herpesviruses (CVSS and CVPF).…”

A Novel Geranylgeranyl Transferase Inhibitor in Combination with Lovastatin Inhibits Proliferation and Induces Autophagy in STS-26T MPNST Cells

“…2Z-GGMP [compound 8; synthesized from 2Z-geranylgeraniol compound 4 in a similar manner to that described for 3-allylfarnesyl monophosphate (Clark et al, 2007)] and 2Z-GGPP [compound 9 (Zahn et al, 2001)] were evaluated against mammalian GGTase I (Hartman et al, 2005) in a modified version of the previously published FTase biochemical continuous fluorescent assay (Reigard et al, 2005). For all assays, the excitation wavelength used was 340 nm, and emission was monitored at 500 nm.…”

Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

“…(10)(11)(12) The sequence context of this cysteine residue determines the enzyme responsible for its modification, with a cysteine within a C-terminal CaaX sequence recognized by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). (11,(13)(14)(15)(16)(17) Alternatively, a cysteine present in a C-terminal CC or CxC motif can be modified by protein geranylgeranyltransferase type II (GGTase-II), also known as Rab GGTase. (18)(19)(20) Cysteine prenylation alters the biophysical properties of proteins in several well-studied cases (e.g.…”

“…Biochemical and cell-based studies, including single amino acid mutations at each position of the CaaX sequence and reengineering of FTase and GGTase-I substrate selectivity, have contributed to the development of rules for prenyltransferase selectivity at each amino acid position within the CaaX sequence. (11,16,17,(39)(40)(41)(42)(43)(44) Crystallographic studies provide a valuable structural context for consideration of FTase and GGTase-I substrate selectivity. (45) In addition to implicating certain FTase and GGTase-I residues as interacting with the CaaX substrate sequence side chains, the structural models of FTase and GGTase-I revealed contacts to the cysteine and terminal carboxylate group of the CaaX sequence that explain the observed preference for the four amino acid CaaX substrate sequence (Figure 1).…”