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

Abstract

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 Ras superfamily. Farnesylation is essential both for normal functioning of these proteins, and for the transforming activity of oncogenic mutants. Consequently FTase is an important target for anti-cancer therapeutics. Several FTase inhibitors are currently undergoing clinical trials for cancer treatment. Here, we present the crystal structure of human FTase, as well as ternary complexes with the TKCVFM hexapeptide substrate, CVFM non-substrate tetrapeptide, and L-739,750 peptidomimetic with either farnesyl diphosphate (FPP), or a nonreactive analogue. These structures reveal the structural mechanism of FTase inhibition. Some CaaX tetrapeptide inhibitors are not farnesylated, and are more effective inhibitors than farnesylated CaaX tetrapeptides. 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 peptide N terminus and the alpha-phosphate of the FPP substrate. Conformational mapping calculations reveal the basis for the sequence specificity in the third position of the CaaX motif that determines whether a tetrapeptide is a substrate or non-substrate. The presence of beta-branched amino acids in this position prevents formation of the non-substrate conformation; all other aliphatic amino acids in this position are predicted to form the non-substrate conformation, provided their N terminus is available to bind to the FPP alpha-phosphate. These results may facilitate further development of FTase inhibitors.

Figure 1

Chemical structures.

Figure 2

Structure of human FTase in complex with the CIFM-derived L-739,750 peptidomimetic and FPP substrate. (A) Overall structure. (B) 6σ F_O-F_C omit electron density for L-739,750. The FPP substrate and zinc ion are included for reference. (C) Residues forming van der Waals interactions with L-739,750, shown in stereo. The N terminus of the peptidomimetic forms an ion pair with an α-phosphate oxygen of the FPP substrate.

Figure 3

Comparison of CaaX non-substrate and substrate conformations. The superpositions are based on all FTase C_α atoms. The same orientation is shown in each panel. (A) CaaX tetrapeptide non-substrate inhibitor binding conformation. L-739,750 peptidomimetic and CVFM tetrapeptide shown with the FPP molecules observed in these two complexes colored yellow (with peptidomimetic) and blue (with CVFM tetrapeptide). Note that the valine to isoleucine substitution at the a₁ position of the Ca₁a₂X motif does not affect the conformation of these two inhibitors, consistent with the orientation of these residues toward bulk solvent. (B) Peptide substrate binding conformation. TKCVFM and the K-Ras4B peptide TKCVIM (18) are both substrates and adopt the same backbone conformation. The N-terminal residue and the lysine side chain of each of these peptides are omitted from the figure for clarity. Also shown are the FPP analogs (Fig. 1), which are colored according to their observed conformations with these peptides. (C) Comparison of the hexapeptide substrate TKCVFM and the non-substrate tetrapeptide inhibitor CVFM. A boxed region highlights the differences between the substrate and non-substrate conformations. (D) Energetically unfavorable steric contact (red spikes) between the C_γ2 methyl group of the isoleucine residue in the a₂ position and the carbonyl oxygen of the a₁ residue when the phenylalanine residue of the CVFM tetrapeptide is replaced with isoleucine. Consequently, a tetrapeptide with a β-branched residue (isoleucine or valine) in the a₂ position cannot adopt the non-substrate binding mode observed for CVFM. These steric contacts were displayed by using the programs probe and reduce (34) in conjunction with the program o (35).

Figure 4

Conformational maps comparing placement of isoleucine (solid line) and phenylalanine (dashed line) in the a₂ position of a tetrapeptide that adopts a non-substrate conformation. The coordinates of the CVFM peptide complex were used as a representative non-substrate conformation. Only the a₁a₂ dipeptide was considered in the calculation. Shown are the contributions to the van der Waals interactions as the φ dihedral angle between the a₁ and a₂ positions is varied. For each sampled value of this angle, the χ₁ dihedral angles of the side chains in the a₁ (valine) and a₂ (isoleucine, valine, phenylalanine, leucine, or alanine) positions were allowed to vary within ±30° of the values observed in the crystal structure, and the minimum local energy was recorded. In the a₂ position, valine resulted in a curve similar to isoleucine, whereas leucine and alanine yielded curves similar to phenylalanine (not shown). The shaded areas indicate the two regions in the vicinity of the non-substrate (φ = −154°, arrow) and substrate (φ = −126°, arrow) conformations, respectively (the conformational map outside these regions is not relevant, because the tetrapeptide is not observed to adopt such conformations in the FTase active site). Conformational maps were generated by using the dezymer program (36, 37).