Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design

Abstract

Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high-resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide substrate, corresponding to successive steps in ordered substrate binding, revealed that the second substrate-binding step is accompanied by motions of a loop in the catalytic site. Re-examination of other FTase structures showed that this motion is conserved. The substrate- and product-binding clefts in the AfFTase active site are wider than in human FTase (hFTase). Widening is a consequence of small shifts in the α-helices that comprise the majority of the FTase structure, which in turn arise from sequence variation in the hydrophobic core of the protein. These structural effects are key features that distinguish fungal FTases from hFTase. Their variation results in differences in steady-state enzyme kinetics and inhibitor interactions and presents opportunities for developing selective anti-fungal drugs by exploiting size differences in the active sites. We illustrate the latter by comparing the interaction of ED5 and Tipifarnib with hFTase and AfFTase. In AfFTase, the wider groove enables ED5 to bind in the presence of FPP, whereas in hFTase it binds only in the absence of substrate. Tipifarnib binds similarly to both enzymes but makes less extensive contacts in AfFTase with consequently weaker binding.

Keywords: FTI; antifungal drugs; crystal structure; enzyme kinetics; farnesyltransferase inhibitors; isoprenoids; lipids; pathogens; posttranslational modification; prenyltransferase; structure-based drug design.

Figure 1

Chemical structures of protein farnesylation substrates, analogs and products. (A) The reaction catalyzed by protein farnesyltransferase on protein substrates bearing the C-terminal CaaX motif. (B) Structure of the isoprenoid analog, farnesyl diphosphate inhibitor II (FPT-II).

Figure 2

Comparison of the tertiary structures of farnesyltransferases from A. fumigatus (AfFTase) and human (hFTase, PDB ID 1TN6). (A) The α (green) and β (magenta) subunits of AfFTase with substrates FPT-II, the pentapeptide sequence KCVVM (yellow sticks), and divalent metal Zn²⁺ (pink sphere). (B) Insertions in the AfFTase α and β subunits (red) alter its Cα backbone relative to the hFTase subunits, resulting in global movements of secondary structures that widen the active site. An interactive view is available in the electronic version of the article. (C) Superposition of the tertiary structures of AfFTase (green) and hFTase (gray) showing an RMSD of 1.8-Å calculated over homologous regions.

Figure 3

Global rearrangements of secondary structures lead to wider active sites in fungal FTases. (A) Schematic representation of the active site funnel and the prenylated product exit groove of protein farnesyltransferase (FTase). The substrates farnesyldiphosphate (FPP, green sticks) from the A. fumigatus FTase (AfFTase) binary complex and the Cys-Val-Val-Met peptide (yellow sticks) from the AfFTase ternary complex with the FPP analog, FPT-II, are shown. FPP and FPT-II bind in essentially the same manner to the isoprenoid binding pocket of AfFTase. The superimposed structure of the displaced farnesylated peptide product (thin gray lines) of hFTase (PDB ID 1KZO) is also shown to highlight the position of the prenylated product exit groove relative to the active site funnel. (B) Surface representation of the active site funnels of protein farnesyltransferase from A. fumigatus (AfFTase), C. neoformans (CnFTase, PDB ID 3Q75), and human (hFTase, PDB ID 1TN6) viewed from the top of the active site funnel. Residues whose atoms form the walls of the active site funnel are shown as sticks and colored green (AfFTase), blue (CnFTase), and pink (hFTase). A black line traces the exit groove tunnel adjacent to the active site funnel. Fungal FTases have wider active site funnels arising from insertions in the α and β subunits relative to hFTase. The FPP analog, FPT-II, and peptide substrates bound in the active site are shown as sticks. An interactive view is available in the electronic version of the article. (C) Widening of the active site funnel causes longer distances between substrate (FPT-II and CVVM peptide in AfFTase, cyan; FPT-II and CNIQ peptide in hFTase (PDB ID 1TN6), gray; and residue atoms in the active site. Selected residues to illustrate the increase in distances are shown in sticks (cyan, AfFTase; gray, hFTase). Distances between atoms are indicated by dashed lines (cyan, AfFTase; gray, hFTase). The positioning of the α helices in the α and β subunits of hFTase result in a narrower active site funnel. The amino acid(s) preceding the Ca₁a₂X cysteine in both structures have been omitted for clarity.

Figure 4

The prenylated product exit groove in the β subunit is highly diverged in sequence and structure among FTases. Comparison of the exit grooves of the protein farnesyltransferase ternary complexes of A. fumigatus (AfFTase, green), C. neoformans (CnFTase, purple PDB ID 3Q75), and human (hFTase, magenta, PDB ID 1TN6) show that the product exit groove in AfFTase and CnFTase are wider than hFTase, with the β3 helix (143-147β) of AfFTase displaced 2-4Å relative to that of hFTase. The bound FPP and displaced farnesylated CVIM peptide (light blue sticks) are modeled using a superimposed structure of the hFTase displaced product complex (PDB ID 1KZO). Amino acid residues that form the walls of the product exit groove are shown as sticks.

Figure 5

A look into the active site of A. fumigatus protein farnesyltransferase (AfFTase). (A) A 2F_o−F_c map contoured at 3.5 σ and calculated to 1.45-Å resolution shows the distorted pentacoordinate geometry of Zn²⁺ coordination. (B) The AfFTase active site showing bound isoprenoid (FPT-II, light green sticks) and peptide (yellow sticks) substrates. Residues that interact with FPT-II (or FPP) by hydrogen bonding (blue dashes) are shown as sticks. The D423β residue putatively coordinates a Mg²⁺ ion during the reaction that is required for efficient catalysis. The Ca₁a₂X peptide (yellow sticks) binds in a fully extended conformation, anchored by coordination with Zn²⁺ (pink sphere) and hydrogen bonding with Q110α (green sticks). Residues that form the surface of the Ca₁a₂X peptide binding site are also shown (purple sticks).

Figure 6

Ligand-induced conformational change in the conserved 4α-5α loop of fungal and mammalian protein farnesyltransferases. (A) In A. fumigatus FTase (AfFTase), the loop conformation in the ternary complex (FPT-II-(replaced by FPP from the AfFTase binary complex) and KCVVVM peptide-bound, green) enables K107α to form a hydrogen bond with the backbone carbonyl of the lysine residue of the KCVVM peptide. Y109α is positioned away from the carboxylate end of the peptide. In the binary complex loop conformation (FPP bound, magenta), the N atom of K107α is moved away from the original position and is unable to form this hydrogen bonding interaction. The conformation of Y109α in this loop conformation clashes with the carboxylate end of the peptide (red arrow). Sequence alignment shows that K107α, Y109α, and Q110α are conserved in mammalian and fungal FTases. An interactive view is available in the electronic version of the article. (B) Similar but smaller ligandinduced loop motions in mammalian FTase. The conformation is shown for different steps in the reaction cycle (bound isoprenoids were omitted in the representations for clarity): apoenzyme (PDB ID 1FT1, gray); AfFTase with bound FPP analog, FPT-II, and the DDPTASACNIQ peptide (PDB ID 1TN6, purple), farnesylated CVLS peptide (PDB ID 2H6F, blue), FPP, and displaced farnesylated CVIM product (PDB ID 1KZO, green). The Cα backbone of 4α-5α loop of hFTase has less flexibility than the corresponding loop in AfFTase (∼1.2 Å shift from apoenzyme to displaced product complex). Residue K164α appears to undergo minimal shift in position; rotamer flipping is observed for Tyr166α and Q167α upon peptide binding (purple arrows).

Figure 7

Binding modes of ethylenediamine-scaffold inhibitor, ED5, and Tipifarnib in A. fumigatus and human protein farnesyltransferases. (A) Inhibitor ED5 contains moieties that bind to the peptide binding site, mobile loop (orange), and the product exit groove. In A. fumigatus farnesyltransferase (AfFTase), ED5 (cyan) binds in the presence of FPP in the active site. In hFTase, ED5 (gray sticks) is competitive with both isoprenoid and peptide substrates, precluding the binding of FPP. In AfFTase, ED5 is competitive with peptide substrate alone. An interactive view is available in the electronic version of the article. (B) The binding mode of tipifarnib in AfFTase (cyan) is conserved with hFTase (gray). In both AfFTase and hFTase, Tipifarnib binds in the presence of FPP (gray, hFTase; cyan, AfFTase). The weaker affinity of Tipifarnib to AfFTase is due to active site widening, as indicated by distances between atoms of the inhibitor and residues that form the active site funnel. Distances are indicated by colored numbers and dashed lines (gray, hFTase; cyan, AfFTase).

Figure 8

Ethylene glycol molecules in the A. fumigatus protein farnesyltransferase (AfFTase) active site suggest routes for inhibitor optimization. The product exit grooves of AfFTase in complex with ED5 (green) and Tipifarnib (magenta) bind ethylene glycol molecules in similar positions (green in ED5 complex, magenta in Tipifarnib complex). An additional ethylene glycol molecule is found in a small cleft in the AfFTase Tipifarnib complex bordered on one side by Y109α, which is not found in a similar tunnel in the AfFTase ED5 complex due to occupancy by the p-benzonitrile moiety of the inhibitor.