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Journal of Lipid Research, Vol. 47, 681-699, April 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Thematic Review

Thematic review series: Lipid Posttranslational Modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I

Kimberly T. Lane and Lorena S. Beese¹

Department of Biochemistry, Duke University Medical Center, Durham, NC 27710

Published, JLR Papers in Press, February 13, 2006.

¹ To whom correspondence should be addressed. e-mail: lsb{at}biochem.duke.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION STRUCTURAL CHARACTERIZATION OF... INHIBITION OF CaaX... STRUCTURAL ANALYSIS OF CaaX... REFERENCES

 
More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation). Prenylated proteins include members of the Ras, Rab, and Rho families, lamins, CENPE and CENPF, and the subunit of many small heterotrimeric G proteins. This modification is catalyzed by the protein prenyltransferases: protein farnesyltransferase (FTase), protein geranylgeranyltransferase type I (GGTase-I), and GGTase-II (or RabGGTase). In this review, we examine the structural biology of FTase and GGTase-I (the CaaX prenyltransferases) to establish a framework for understanding the molecular basis of substrate specificity and mechanism. These enzymes have been identified in a number of species, including mammals, fungi, plants, and protists. Prenyltransferase structures include complexes that represent the major steps along the reaction path, as well as a number of complexes with clinically relevant inhibitors. Such complexes may assist in the design of inhibitors that could lead to treatments for cancer, viral infection, and a number of deadly parasitic diseases.

Supplementary key words prenyltransferase • isoprenoid • Ras • G protein • cancer target • drug design • farnesyltransferase inhibitor • crystal structure

Abbreviations: FPP, farnesyl diphosphate; FTase, protein farnesyltransferase; FTI, farnesyltransferase inhibitor; GGPP, geranylgeranyl diphosphate; GGTase-I, geranylgeranyltransferase type I; GTI, geranylgeranyltransferase inhibitor; PDB ID, Protein Data Bank identifier

	INTRODUCTION

TOP ABSTRACT INTRODUCTION STRUCTURAL CHARACTERIZATION OF... INHIBITION OF CaaX... STRUCTURAL ANALYSIS OF CaaX... REFERENCES

 
More than 100 proteins necessary for eukaryotic cell growth, differentiation, and morphology require posttranslational modification by the covalent attachment of an isoprenoid lipid (prenylation) (1). This modification is catalyzed by three protein prenyltransferases: protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I), collectively termed the CaaX prenyltransferases, as well as protein GGTase-II (or RabGGTase) [reviewed in this series in (2)], whose substrates are limited to members of the Rab subfamily of G proteins. FTase and GGTase-I transfer a 15 or 20 carbon isoprenoid [donated by farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP)], respectively, to the cysteine of a C-terminal CaaX motif, defined by a cysteine (C) residue, followed by two small, generally aliphatic (a) residues, and the X residue, which contributes significantly to specificity (Fig. 1 ) (3–9). Kinetic assays and analysis of lipidated CaaX proteins purified from the cell demonstrate the general preference of FTase for methionine, serine, glutamine, or alanine and the preference of GGTase-I for leucine or phenylalanine in the X position. Here, we review the structural biology of FTase and GGTase-I; the biochemical properties of these enzymes have been reviewed extensively elsewhere (1, 10–15).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Biochemistry of the CaaX prenyltransferases. Ala, alanine; Gln, glutamine; Leu, leucine; Met, methionine; Phe, phenylalanine; Ser, serine.

View larger version (44K): [in this window] [in a new window]   Fig. 2. CaaX prenylation and the eukaryotic cell cycle. The prenylation of CaaX proteins (shown here is Ras, in yellow, as an example) by protein prenyltransferases (red and blue ribbon diagram) and further processing by Rce1 (purple) and Icmt (green) are depicted. Also shown is the cholesterol biosynthesis pathway [adapted from (178)], from which the isoprenoid diphosphate substrates farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are derived. A simplified depiction of the Ras signaling pathway, after its insertion into the membrane via its lipid tail, is also illustrated [adapted from (179)]. EGF, epidermal growth factor; GAP, GTPase activating protein; GEF, guanine exchange factor; HMG CoA, 3-hydroxyl-3-methylglutaryl Coenzyme A; Icmt, isoprenylcysteine carboxyl methyltransferase; MEK, mitogen-activated protein (MAP) or extracellular signal-regulated kinase (ERK) kinase; Mnk, MAP kinase-interacting kinase; Rce1, Ras- and a-factor-converting enzyme; RTK, receptor tyrosine kinase.

Since the initial discovery of farnesylated fungal proteins, many more proteins have been demonstrated to contain a prenyl modification, including >100 human proteins. These include members of the Ras (39–42), Rho (43–47), Rac (48), Rap (49, 50), and Rab (51, 52) families, the subunit of heterotrimeric G proteins (53, 54), centromeric proteins (55), and many other proteins involved in a variety of cell signaling pathways controlling the cell cycle and apoptosis (56, 57) as well as glycogen metabolism (58), cellular framework (59–61), and visual signal transduction (53, 62–64). Defects in these proteins are associated with a number of human diseases, including Batten disease (65), autosomal recessive retinitis pigmentosa and autosomal dominant retinitis pigmentosa (66–68), Canavan disease (69), Emery-Dreifuss muscular dystrophy type 2 (70–72), and a variety of human cancers. Some of the most studied CaaX proteins are members of the Ras family, mutations in which occur in 20–30% of all human tumors, making the CaaX prenyltransferases targets for inhibition in cancer therapeutics (73, 74) (see below).

	STRUCTURAL CHARACTERIZATION OF THE CaaX PRENYLTRANSFERASES

TOP ABSTRACT INTRODUCTION STRUCTURAL CHARACTERIZATION OF... INHIBITION OF CaaX... STRUCTURAL ANALYSIS OF CaaX... REFERENCES

 
Over the past decade, high-resolution crystal structures of FTase and GGTase-I complexed with various substrates, products, and inhibitors have advanced our understanding of these enzymes. Structures representing each of the major steps along the reaction pathway of CaaX prenyltransferases have been determined (Fig. 3 ) (75, 76). Kinetic studies suggest an ordered binding mechanism in FTase, in which FPP binds to the apo enzyme first, followed by the CaaX substrate (77–80). The release of farnesylated product is the rate-limiting step of the reaction and occurs only in the presence of excess of either substrate, particularly isoprenoid diphosphate (3, 8, 78, 81, 82). This reaction cycle effectively maintains FTase in a substrate- or product-bound complex at all times. Although the GGTase-I reaction has not been studied in such detail, available evidence indicates that it proceeds through a similar pathway (83, 84).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Structures along the protein farnesyltransferase (FTase) reaction path. Crystallographic studies (75, 85, 95, 99) have produced structures representing the major steps along this path. In each of these complexes, the enzyme acts as a rigid scaffold; therefore, for clarity, only the substrates and products are shown as they bind in the active site. The path begins with the unliganded (apo) enzyme (0) [Protein Data Bank identifier (PDB ID) 1FT1], with the subunit shown in red and the ß subunit in blue. The FPP molecule binds to form a binary substrate complex (1) (PDB ID 1FT2), followed by binding of the CaaX substrate to form a ternary substrate complex (2) (PDB ID 1D8D). The resulting farnesylated product remains bound in the active site (3) (1KZP). Excess substrate, particularly FPP, facilitates product displacement, demonstrated by a complex in which the new FPP molecule and the partially displaced product are bound simultaneously (4) (1KZO). The double dagger symbol indicates a modeled transition state along the reaction coordinate between 2 and 3 (see Fig. 12 for a more detailed view of the transition state). Throughout this figure, the isoprenoid is shown in blue, the CaaX in yellow, and the catalytic zinc ion in magenta. Also shown are the kinetic parameters determined for this reaction (3, 8, 77–81). Reproduced with permission from (75).

View larger version (19K): [in this window] [in a new window]   Fig. 12. Proposed transition state model for the CaaX prenyltransferases. The scissile phosphoether bond between the diphosphate and prenyl groups and the nascent thioether bond between the CaaX cysteine and the prenyl group are shown as black dotted lines. Hydrogen bonds predicted to stabilize the phosphate in the transition state are shown as red dotted lines (residues of FTase are labeled in blue, GGTase-I in red). In this model, the magnesium ion, in turn coordinated by D352ß, in FTase is replaced by K311ß in GGTase-I. Lys, lysine.

View larger version (61K): [in this window] [in a new window]   Fig. 4. Structures of the CaaX prenyltransferases. Overall structures of FTase (A) and geranylgeranyltransferase type I (GGTase-I) (B), with the subunit shown in red, the ß subunit in blue and yellow, respectively, and the catalytic zinc ion in magenta. C: Superposition of FTase (blue) and GGTase-I (yellow) demonstrates the structural homology of the ß subunit of these enzymes.

Although the ß subunits of FTase and GGTase-I share only 25% sequence identity, they have very similar structures, consisting of 14 helices in FTase and 13 in GGTase-I. Twelve of the helices are folded into an - barrel (Fig. 4C). Six parallel helices (3ß, 5ß, 7ß, 9ß, 11ß, and 13ß) create the core of the barrel. The other six helices (2ß, 4ß, 6ß, 8ß, 10ß, and 12ß) are parallel with one another and antiparallel with respect to the core helices and form the outside of the barrel. In both enzymes, one end of this barrel is blocked by a loop containing the C-terminal residues of the ß subunit, whereas the opposite end is open to the solvent. This arrangement creates a deep, funnel-shaped cavity in the center of the barrel, with an approximate inner diameter of 15 Å and a depth of 14 Å. This cavity is hydrophobic in nature and lined with a number of conserved aromatic residues. The active sites of both FTase and GGTase-I are located within this cavity.

The and ß subunits form an extensive interface, burying >3,000 Ų, or 20%, of accessible surface area of each subunit (76, 85). The /ß interfaces of both FTase and GGTase-I have lower hydrophobic character than is observed in most subunit interfaces (88). The unusually high polar/charged residue content results in nearly double the typical number of hydrogen bonds.

Zinc binding site
Both FTase and GGTase-I are zinc metalloenzymes, binding one Zn²⁺ ion per protein dimer (89, 90). Crystal structures of FTase and GGTase-I clearly indicate the presence of a single Zn²⁺ ion bound to the ß subunit near the subunit interface (76, 85). In both enzyme structures, the Zn²⁺ ion is coordinated by three strictly conserved residues: D297ß, C299ß, and H362ß in FTase and D269ß, C271ß, and H321ß in GGTase-I. Mutagenesis studies performed on FTase are consistent with Zn²⁺ coordination by residues D297ß, C299ß, and H362ß (91–93) and additionally implicate D359ß in the binding of zinc (92). Crystal structures of FTase (85, 87) reveal a stabilizing hydrogen bond between D359ß and the zinc ligand H362ß, consistent with a secondary coordination shell effect exerted by the former. A similar interaction is observed in GGTase-I (76).

In crystal structures of FTase (94–97) and GGTase-I (76, 97), the CaaX cysteine thiol(ate/ether) is observed coordinating the zinc, consistent with biochemical studies that indicate that the Zn²⁺ ion is required for catalytic activity and that it coordinates the cysteine thiol of the CaaX substrate (12, 19, 32, 81, 89). Comparison of zinc coordination from high-resolution substrate [1.8 Å (97)] and product [1.65 Å (K. L. Terry et al., unpublished observations)] complexes indicates that the CaaX cysteine thiol(ate/ether) forms a short (2.3 Å) interaction with the zinc ion in substrate complexes and a slighter longer (2.6 Å) interaction in the product complex (Fig. 5 ). The complete zinc coordination sphere for the substrate complex has four close interactions (D297ß, 2.0 Å; C299ß, 2.3 Å; H362ß, 2.1 Å; CaaX cysteine, 2.3 Å) and one longer interaction (D297ß, 2.6 Å; making this residue a bidentate ligand). The prenylated peptide product complex has a similar coordination sphere (D297ß, 2.0 and 2.4 Å; C299ß, 2.3 Å; H362ß, 2.2 Å; CaaX prenyl cysteine, 2.6 Å). Extended X-ray absorption fine structure (EXAFS) data (98) confirm the short interactions with the zinc ion seen in these crystal structures. Longer interaction distances, such as the bidentate interaction of D297ß, in substrate complexes appear to be sufficiently weak to not be detected by this technique.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Zinc coordination in FTase. The catalytic zinc ion (magenta) is shown in a ternary substrate complex (A) (PDB ID 1TN6) and a farnesylated peptide-product complex (B), interacting with three enzyme residues (D297ß, C299ß, and H362ß) as well as the cysteine (Cys) of the CaaX motif. Note the longer interaction distance between the Zn2+ ion and the CaaX cysteine in the product complex.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Isoprenoid diphosphate binding in the CaaX prenyltransferases. FPP (yellow) in FTase (PDB ID 1FT2) (A) and GGPP (purple) in GGTase-I (PDB ID 1N4P) (B) bind along one side of the hydrophobic pocket. Reproduced with permission from (99).

Superposition of FTase and GGTase-I in binary isoprenoid substrate complexes by aligning all enzyme C atoms illustrates the similarity of binding of FPP and GGPP in their respective enzymes (Fig. 7A ) (76). There is a slight divergence in the binding of the first isoprene unit, but the second and third units are essentially indistinguishable. The binding sites for GGPP in GGTase-I and FPP in FTase are very similar, constituting a cavity lined with conserved aromatic residues. The primary differences are at the bottom of this cavity, where the fourth isoprene unit of GGPP binds in GGTase-I. Here, FTase has the bulky tryptophan and tyrosine residues (W102ß and Y365ß), whereas the corresponding positions in GGTase-I (and RabGGTase) are occupied by the smaller residues threonine and phenylalanine to accommodate the fourth isoprene unit of the GGPP molecule, which cannot fit in FTase. Therefore, FTase and GGTase-I can function as simple length-discriminating molecular rulers. This hypothesis was tested by introducing mutations into FTase that are predicted to convert it to a geranylgeranyltransferase (76). A mutation at the W102ß residue of FTase to a threonine (the corresponding residue in GGTase-I) (W102T mutant) was created. Steady-state activity assays indicated that this single mutation results in an FTase enzyme greatly preferring GGPP over FPP as its isoprenoid substrate without significantly altering CaaX sequence specificity (Fig. 7B).

View larger version (17K): [in this window] [in a new window]   Fig. 7. CaaX prenyltransferase isoprenoid diphosphate substrate binding pocket. A: Comparison of isoprenoid binding in FTase (PDB ID 1KZ0) and GGTase-I (PDB ID 1N4P). In FTase (red), the larger tryptophan (Trp) fills the space where the fourth isoprene binds in GGTase-I (blue) and is one of the primary determinants of isoprenoid specificity. Thr, threonine; Tyr, tyrosine. B: Altered substrate specificity of the W102T mutant. Prenylation reactions assayed the activity of human wild-type FTase, wild-type GGTase-I, and the W102T mutant with four substrate combinations: FPP + Ras-CVLS (blue), FPP + Ras-CVLL (green), GGPP + Ras-CVLS (yellow), and GGPP + Ras-CVLL (red). Reproduced with permission from (76).

In each of these structures, the isoprenoid forms a large part of the CaaX sequence binding surface, with its second and third isoprene (and fourth, in the GGPP analog) units in direct van der Waals contact with the CaaX motif, particularly the a₂ and X residues (Fig. 8A , B). The peptide substrate sandwiches the isoprenoid against the wall of the hydrophobic cavity, burying 115 and 140 Ų (in FTase and GGTase-I, respectively) of accessible isoprenoid diphosphate analog surface area and completely sequestering the third isoprene unit from the bulk solvent. The location and conformation of the nonreactive isoprenoid analogs used in these studies are similar to those observed in the binary complex with FPP and GGPP. Furthermore, the enzyme residues that interact with the isoprenoid diphosphate substrate molecule were also observed to interact with the analogs. These structures, therefore, are consistent with the ordered binding pattern observed by kinetics in which isoprenoid diphosphate binding precedes peptide binding (77, 78).

View larger version (39K): [in this window] [in a new window]   Fig. 8. CaaX substrate binding in the CaaX prenyltransferases. CaaX substrates bind in FTase (A) and GGTase-I (B) opposite the isoprenoid diphosphate substrate (PDB IDs 1D81 and 1N4Q). Also shown are the residues of the enzymes interacting with the CaaX motif, as well as the isoprenoid diphosphate analog (purple) and the catalytic zinc (dark blue in FTase, magenta in GGTase-I). Asp, aspartic acid; Glu, glutamic acid; His, histidine. Reproduced with permission from (76, 95).

Further insight into the role of zinc in peptide substrate binding is derived from a crystal structure of zinc-depleted rat FTase with bound FPP analog and a K-Ras4B-derived peptide (PDB ID 1D8E) (95). The overall structure of the enzyme is essentially identical to the other FTase structures, proving that zinc is not required to maintain the proper three-dimensional fold of FTase. Furthermore, the conformations of the zinc ligands (D297ß, C299ß, and H362ß) remain largely undisturbed. Both the FPP analog and peptide are observed bound in the active site without zinc. The FPP analog conformation is unchanged from the other ternary complexes, supporting the observation that zinc is not required for FPP binding (89). In this complex, the a₂ and X residues of the CaaX peptide bind in the same manner as in the presence of zinc, whereas the cysteine and valine residues bind much differently, forming a ß-turn. The cysteine thiol(ate) has shifted 9 Å from its position when in coordination of the zinc ion. This altered CaaX binding conformation of the peptide in the absence of zinc indicates that it is the zinc-thiol(ate) interaction that anchors the cysteine residue, holding the peptide in an extended conformation. This observation is consistent with the finding that peptide affinity is greatly enhanced in the zinc metalloenzyme (89). This zinc-depleted structure also indicates that, although not strictly required for peptide binding, the zinc ion is required both to orient the cysteine thiol(ate) for catalysis and to stabilize a productive peptide substrate conformation.

CaaX protein substrate specificity
FTase and GGTase-I possess distinct but overlapping protein substrate specificities that are determined by the degenerate Ca₁a₂X sequence motif, recognizing a number of protein substrates with a variety of sequences (3–9). To identify the basis for peptide selectivity by the CaaX prenyltransferases, structures were determined of FTase and GGTase-I complexed with the appropriate isoprenoid diphosphate analogs and a collection of specific and cross-reactive peptide substrates, including sequences derived from the C terminus of the oncoproteins K-Ras4B (CVIM; PDB ID 1TNO in GGTase-I), H-Ras (CVLS; PDB ID 1TN8 in FTase), and TC21 (CVIF; PDB IDs 1TN7 in FTase and 1TNB in GGTase-I), the signal transduction proteins Rap2a (CNIQ; PDB ID 1TN6 in FTase), RhoB (CKVL; PDB ID 1TNU in GGTase-I), and Cdc42 (CVLL; PDB ID 1TNZ in GGTase-I), and the heterotrimeric G protein ₂ subunit (CAIL; PDB ID 1TNY in GGTase-I) (97).

A superposition of these ternary complexes by aligning all C atoms (Fig. 9 ) reveals that the CaaX portion of all protein substrates adopts a common binding mode in the FTase and GGTase-I active sites and suggests a series of rules that define peptide substrate selectivity for the CaaX prenyltransferases. The presence of two fixed anchor points within the recognition motif (cysteine and C terminus) that make specific interactions with the enzyme discriminates against peptides that are either too long or too short (i.e., only tetrapeptides) or that lack a cysteine at the correct position. The a₁ residue is solvent-exposed and can accommodate any amino acid, as observed in solution studies (6, 76, 94, 95). Both the a₂ and X positions are buried in the active site; consequently, they are the major determinants of peptide selectivity through steric and electrostatic complementarity between the amino acid side chains and enzyme residues.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Comparison of Ca1a2X substrate binding in FTase and GGTase-I. Superpositions of four FTase substrate complexes (left panel) and three GGTase-I substrate complexes (right panel) demonstrate that cognate and cross-reactive peptides adopt a common binding mode (PDB IDs 1D81, 1TN6, 1TN7, 1TN8, 1TNO, 1TNU, and 1TNB). In FTase, however, the C-terminal phenylalanine residue of TC21 binds in a different pocket than the C-terminal methionine, glutamine, and serine residues. Reproduced with permission from (97).

View larger version (38K): [in this window] [in a new window]   Fig. 10. Comparison of the a2 binding site in FTase and GGTase-I. Superposition of FTase and GGTase-I shows residues that interact with the a2 residue of the Ca1a2X motif (PDB IDs 1D81 and 1N4Q). Regions of the FTase ternary complex forming the a2 binding site (enzyme residues W102ß, W106ß, and Y361ß and isoprene 3) are colored red. Corresponding regions of the GGTase-I ternary complex (enzyme residues T49ß, F53ß, and L321ß, isoprenes 3 and 4, and substrate peptide X residue) are colored blue. Portions of the FPP and GGPP analogs (gray) and of the CVIM (pink) and CVIL (light blue) peptides not forming the a2 site are also shown. Reproduced with permission from (97).

Together, these structures outline a series of rules that govern substrate peptide selectivity. These rules were used to identify potential protein substrates within the human genome, including a number of possible CaaX prenyltransferase substrates not identified previously.

Product formation
In both FTase and GGTase-I, the structures of the product complex (complex 3 in Fig. 3) (PDB IDs 1KZP and 1N4R) and ternary substrate complexes are virtually identical (75). There are no observed changes in the polypeptide backbone; differences are confined to changes in side chain rotamers of those active site residues that interact with the diphosphate moiety of the isoprenoid diphosphate substrate and are a consequence of the pyrophosphate product release. Equally, the peptide exhibits no appreciable differences between its reactant and product structures (Fig. 11A ). The isoprenoid, however, moves toward the peptide in the product structure, with the C₁ atom nearly 7 Å closer to the Zn²⁺ ion compared with its position as a substrate. This motion is localized, as the third (and fourth, in the geranylgeranyl moiety) isoprene unit retains its substrate binding position; only the first and second isoprene units shift, and they do so by rotating through the second isoprene unit. These results suggest that only a portion of the isoprenoid moves substantially during catalysis.

View larger version (29K): [in this window] [in a new window]   Fig. 11. Comparison of the binding of substrates and products in the CaaX prenyltransferases. A: In FTase, FPP (dark blue) rotates through the second isoprene unit to close the distance between the C1 atom and the CaaX substrate (light blue) (PDB ID 1D8D) to form a farnesylated peptide product (brown and yellow) (PDB ID 1KZP). Ile, isoleucine; Val, valine. B: Simultaneous binding of FPP and translocated product in FTase (PDB ID 1KZO). FPP (blue) binds along one side of the active site. The CaaX portion of the translocated product (yellow) no longer coordinates the zinc ion (magenta), making a type I-ß turn, placing the farnesyl portion (brown) of the product in a conserved "exit groove" (residues shown in cyan). Reproduced with permission from (75).

Implications of substrate-mediated product release
Unlike FTase and GGTase-I, RabGGTase is a processive enzyme, adding two prenyl groups to its Rab substrates (52) via a process that appears to proceed without dissociation of the monoprenylated intermediate (109) [reviewed in this series in (2)]. Biochemical and structural studies indicate that, like FTase and GGTase-I, RabGGTase has only one prenyl diphosphate binding site (101, 110). The structure of RabGGTase (PDB ID 1DCE) revealed a striking 7.5 Å diameter hydrophobic tunnel, originating from a shallow groove analogous to the exit groove in FTase and GGTase-I and extending through the ß subunit (101, 110). The first geranylgeranyl isoprenoid attached to Rab may be sequestered in this tunnel after translocation of the monoprenylated product from the site of catalysis, which may be associated with the binding of fresh GGPP substrate, as observed for FTase and GGTase-I (109, 111). This translocation would allow the unmodified cysteine residue to interact with the zinc ion in preparation for the second catalytic reaction. This mechanism would explain why RabGGTase preferentially modifies Rab substrates that have one geranylgeranyl group already attached (52, 109). In addition, the potential to insert different lengths of the geranylgeranyl moiety through the tunnel may explain how RabGGTase is able to modify cysteine residues at a variety of positions within a few residues of the C termini of Rab substrates.

Additionally, this unusual substrate-mediated product release may have a significant biological role. We propose that it provides a mechanism for the regulated handover of the prenylated protein product to the next step in this processing pathway (76). In the CaaX prenyltransferases, the prenylated product remains tightly bound; therefore, it is shielded from the cytoplasm, preventing aggregation or association with an incorrect membrane compartment. Only upon binding of an additional isoprenoid diphosphate molecule is the product isoprenoid displaced into the exit groove, presenting the product for delivery to the next processing step. The mechanism for this handover is analogous to one of the roles of Rab escort protein (REP), which escorts RabGGTase products to their final destination (112), and is similar to that of the GDI proteins, which extract prenylated G proteins such as Rho from membranes by binding the isoprenoid moiety in a hydrophobic pocket and transporting them throughout the cell (47).

Transition state model
The chemical mechanism and nature of the transition state is still an area of active investigation. We have developed a transition state model for catalysis by CaaX prenyltransferases consistent with the observed structural and kinetic information (Fig. 12 ) (75, 76). The proposed model retains elements of both the postulated electrophilic and nucleophilic components of the reaction mechanism (80, 113, 114). In this model, the CaaX motif binds as observed in the ternary substrate complex, coordinating the Zn²⁺ ion, whereas the isoprenoid diphosphate rotates through its second isoprene unit to bring the C₁ atom into alignment for thioether bond formation, maintaining the binding conformation of the ß-phosphate in its substrate conformation. In this reaction, the isoprenoid C₁ atom undergoes inversion of configuration during the reaction (115–117). The developing negative charge on the diphosphate moiety (localized mainly on the -phosphate) is stabilized by several interactions with the enzyme, particularly residues K164 and a ß subunit tyrosine (Y300ß in FTase, Y272ß in GGTase-I). Kinetic studies demonstrated that FTase, but not GGTase-I, requires millimolar levels of Mg²⁺ for full catalytic efficiency (19, 89). It has been hypothesized that the Mg²⁺ ion required by FTase stabilizes the developing negative charge on the diphosphate as the bond breaks between the -phosphate and the C₁ atom of the farnesyl group. Structural studies suggest that Mg²⁺ is coordinated by residue D352ß of the enzyme and the FPP diphosphate moiety (75). Sequence alignments reveal that in GGTase-I, a lysine occupies the position corresponding to D352ß in FTase. Superposition of the structures of FTase (75, 85, 95, 96, 99) and GGTase-I (76) shows that this lysine residue adopts a conformation that positions the positively charged side chain N at the site of Mg²⁺ in FTase, allowing it to stabilize the diphosphate group. Mutagenesis studies (118, 119) indicate that Mg²⁺ sensitivity is dependent on these two residues; a lysine at 352ß of FTase abolishes Mg²⁺ dependence, whereas an aspartate at 311ß in GGTase-I introduces Mg²⁺ dependence.

	INHIBITION OF CaaX PRENYLTRANSFERASE ACTIVITY

TOP ABSTRACT INTRODUCTION STRUCTURAL CHARACTERIZATION OF... INHIBITION OF CaaX... STRUCTURAL ANALYSIS OF CaaX... REFERENCES

 
Three major classes of farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase inhibitors (GTIs) have been identified: isoprenoid diphosphate-derived, peptide-competitive (peptidomimetic and nonpeptidomimetic), and bisubstrate-mimicking [reviewed in this series in (120)]. Hundreds of compounds have been discovered or developed as inhibitors of either FTase or GGTase-I (or both); many of these have clinical potential as anticancer (121–125), antiparasitic (29, 126–128), antifungal (129–131), and antiviral (132–134) therapeutic agents. Some of these have been characterized by X-ray crystallographic studies, revealing a molecular basis for their inhibitory activities.

Isoprenoid diphosphate-derived inhibitors
A number of FPP and GGPP analogs have been developed that are competitive inhibitors of FTase and GGTase-I, respectively (135–141). Systematic structure-activity analysis of a series of FPP-based inhibitor molecules indicated that the most potent inhibitors retain a hydrophobic farnesyl group and a negatively charged moiety mimicking the diphosphate (138), consistent with the interactions observed previously between the enzyme and the terminal phosphate in the FTase and GGTase-I complexes (75, 76, 94–97, 99, 142, 143). That study also revealed the dramatic effect of hydrophobic chain length on FTase activity, because elongation of the farnesyl group by a single carbon resulted in a decrease in IC₅₀ of >200-fold, consistent with the structure-derived ruler hypothesis.

Several isoprenoid analog inhibitors have been characterized in crystal structures of various complexes (76, 94–97, 144, 145). Some of these inhibitors are described in this review (see above), including the FPP analogs farnesyl protein transferase inhibitor II (95–97) and -hydroxyfarnesylphosphonic acid (94) and the GGPP analog 3'-aza GGPP (76, 97). In each of these structures, the isoprenoid analogs bind in a manner similar to their substrate counterparts, interacting extensively with both the enzyme and the CaaX substrate. These inhibitors continue to be invaluable in the structural characterization of FTase and GGTase-I; they bind much like the isoprenoid diphosphate substrates but will not react with the peptide, allowing the crystallization of ternary substrate complexes.

Peptide-competitive inhibitors
Peptidomimetics Initial characterization of FTase demonstrated that this enzyme is inhibited by short Ca₁a₂X peptides (3, 4, 6). The most effective inhibitory peptides contain a nonpolar aliphatic or aromatic amino acid in the a₁ or a₂ position, particularly the latter, although some of these peptides can also serve as FTase substrates. Inclusion of an aromatic residue such as phenylalanine in the a₂ position eliminates prenylation, however, resulting in a purely competitive inhibitor with respect to the peptide. Interestingly, the removal or substitution of the amino group on the cysteine of a tetrapeptide inhibitor restores catalytic activity (4, 7). Because these molecules are peptides, they have three significant disadvantages for potential in vivo activity: poor cellular uptake, rapid degradation, and FTase farnesylation (in some cases), which results in depletion of the inhibitor. To overcome these disadvantages, a number of peptidomimetic inhibitors have been developed and characterized (96, 121, 146–150).

The mechanism for tetrapeptide inhibition was studied using the crystal structures of FTase complexed with the nonsubstrate tetrapeptide CVFM (PDB ID 1JCR), the substrate hexapeptide TKCVFM (PDB ID 1JCS), and the peptidomimetic inhibitor L-739,750 (PDB ID 1JCQ), developed by Merck Research Laboratories (96). This inhibitor compound, mimicking the tetrapeptide CIFM, is the metabolic product of the prodrug L-744,832; administration of this prodrug in rats resulted in tumor regression without any observed systemic toxicity (121). Each of these peptides/peptidomimetics bind in the peptide binding site, anchored by cysteine coordination of the catalytic zinc ion and hydrogen bonds (both direct and water-mediated) between the C terminus and the enzyme. The two inhibitors (the nonsubstrate CVFM peptide and L-739,750) adopt a similar conformation, in which there is an 3 Å distance shift of the inhibitor cysteine C atom relative to the substrate, although the cysteine still coordinates the zinc ion (Fig. 13A , C). This alternative conformation is stabilized by a hydrogen bond between the N terminus of the inhibitor and an oxygen on the -phosphate of the FPP molecule, as well as by hydrophobic interactions between the phenylalanine-derived side chain of the inhibitor and several aromatic residues of the enzyme. In both complexes, the inhibitor binds in the space between the isoprenoid and the zinc ion and interferes with the movement of the isoprenoid during the reaction, preventing bond formation. The substrate TKCVFM hexapeptide, however, adopts the same conformation as the K-Ras4B-derived TKCVIM peptide in an analogous complex (Fig. 13B). In each of these complexes, the side chain in the a₂ position adopts essentially the same conformation; this conformation places steric constraints on the backbone angle between the a₁ and a₂ residues. ß-Branched amino acids (isoleucine and valine) in the a₂ position form an unfavorable steric interaction between their C₂ methyl group and the carbonyl oxygen of the a₁ residue in the nonsubstrate backbone conformation adopted by the CVFM peptide (Fig. 13D). Therefore, it was predicted that, of the CaaX cognate tetrapeptides, only peptides with branched residues (i.e., isoleucine or valine) in the a₂ position are substrates, whereas peptides with other nonbranched aliphatic amino acids are poor substrates or nonsubstrates (provided that the N terminus is not blocked).

View larger version (47K): [in this window] [in a new window]   Fig. 13. Comparison of CaaX nonsubstrate and substrate conformations. A: CaaX tetrapeptide nonsubstrate inhibitor binding conformation. L-739,750 peptidomimetic (PDB ID 1JCQ) and CVFM tetrapeptide (PDB ID 1JCR) are shown with the FPP molecules observed in these two complexes colored yellow (with peptidomimetic) and blue (with CVFM). B: Peptide substrate binding conformation. TKCVFM (PDB ID 1JCS) and the K-Ras4B-derived peptide TKCVIM (PDB ID 1D8D) 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 here for clarity. Also shown are the FPP analogs, which are colored according to their observed conformations with these peptides. C: Comparison of the hexapeptide substrate TKCVFM and the nonsubstrate tetrapeptide inhibitor CVFM. A boxed region highlights the differences between the substrate and nonsubstrate conformations. D: Energetically unfavorable steric contact (red spikes) between the C2 methyl group of the isoleucine residue in the a2 position and the carbonyl oxygen of the a1 residue when the phenylalanine of the CVFM tetrapeptide is replaced with isoleucine. Consequently, a tetrapeptide with a ß-branched residue (isoleucine or valine) in the a2 position cannot adopt the nonsubstrate binding mode observed for CVFM. Reproduced with permission from (96).

The structures of FTase complexed with FPP (or an FPP analog) and each of the inhibitors R115777 (Janssen; PDB ID 1SA4) (142), BMS-214662 (Bristol-Myers Squibb; PDB ID 1SA5) (142), and ABT-839 (Abbott Laboratories; PDB ID 1N94; one of several Abbott compounds with determined structures) (161) have been determined. All three inhibitors bind in the FTase peptide binding site, overlapping with the binding of the Ca₁a₂ portion of the substrate Ca₁a₂X peptide (Fig. 14 ). R11577 and BMS-214662 coordinate the catalytic Zn²⁺ ion via an imidazole group; however, ABT-839 does not. ABT-839 is also unique in that it possesses a methionine mimic at one end of the molecule that binds as in the substrate complex (95), making it the only nonpeptidomimetic inhibitor described here that extends into the X residue binding site.

View larger version (38K): [in this window] [in a new window]   Fig. 14. FTase inhibitors R115777, BMS-214662, and ABT-839. Ribbon cartoons of FTase complexed with FPP or FPP analog -hydroxyfarnesylphosphonic acid (-HFP)(purple) and the inhibitor molecules (orange) R115777 (A), BMS-214662 (B), and ABT-839 (C) shown in the same orientation (PDB IDs 1SA4, 1SA5, and 1N94, respectively). Each inhibitor is shown in comparison with the K-Ras4B-derived peptide CVIM (gray) (PDB ID 1D8D). Also shown is the Zn2+ ion (magenta), coordinated by the imidizole group of R115777 and BMS-214662.

View larger version (47K): [in this window] [in a new window]   Fig. 15. FTase inhibitor SCH66336. Ribbon cartoons of FTase complexed with FPP (purple) and the inhibitor SCH66336 (orange) (PDB ID 1O5M). The inhibitor is shown in comparison with (A) the K-Ras4B-derived peptide CVIM (dark gray) (PDB ID 1D8D) and (B) the partially displaced peptide product (light gray) (PDB ID 1KZO). Also shown is the Zn2+ ion (magenta).

View larger version (51K): [in this window] [in a new window]   Fig. 16. L-778,123 binding in FTase and GGTase-I suggests a molecular mechanism for inhibitor selectivity. Superposition of FTase (blue) and GGTase-I (red) complexes illustrates the binding modes of L-778,123 in each enzyme (PDB IDs 1S63 and 1S64, respectively). Only the residues that contact the a2 residue of the Ca1a2X peptide, the "a2 binding site," and a conserved arginine (Arg) residue are shown. Aromatic stacking interactions are not possible between L-778,123 and the GGTase-I a2 binding site, encouraging the inhibitor to adopt a lipid-competitive binding mode that permits the formation of a hydrogen bond. Reproduced with permission from (143).

	STRUCTURAL ANALYSIS OF CaaX PRENYLTRANSFERASE AND HUMAN THERAPEUTICS

TOP ABSTRACT INTRODUCTION STRUCTURAL CHARACTERIZATION OF... INHIBITION OF CaaX... STRUCTURAL ANALYSIS OF CaaX... REFERENCES

 
Since their discovery, FTase and GGTase-I have been targets for cancer therapeutics (73, 74). To this end, a number of FTIs and GTIs have been studied kinetically, structurally, and clinically (reviewed in this series in 120). More recent studies have begun to illustrate the potential of FTIs in the treatment of infection by pathogenic microorganisms, including Plasmodium falciparum (the causative agent of malaria) (29, 128), Trypanosoma brucei (African sleeping sickness) (126, 173), Trypanosoma cruzi (Chagas disease) (35), and Leishmania mexicana and Leishmania major (leishmaniasis) (35, 127) (reviewed in this series in 174). Another potential application of FTIs and GTIs is as a new class of antifungal and antiviral agents. Candida albicans and Cryptococcus neoformans (opportunistic fungal pathogens causing life-threatening infections in many immunocompromised patients with AIDS) (24, 25, 175, 176) and viruses (through the use of host prenyltransferase enzymes), including hepatitis C virus and hepatitis delta virus, appear to require prenylation for viability (25, 129, 132, 133).

Although structural characterization of mammalian FTase and GGTase-I has advanced our understanding of these enzymes, there is no structural information available for any of the protein prenyltransferases from human pathogens. FTIs are attractive drug candidates to develop for the treatment of parasitic, fungal, and viral infections because the chemistry, pharmacokinetics, and toxicity of a number of these compounds have been established in humans. Using this "piggy-back" approach to drug development bypasses many of the hurdles involved in designing drugs de novo (177). Structural studies of the protein prenyltransferases from human pathogens would facilitate the design of potent, highly selective inhibitors.

	ACKNOWLEDGMENTS

 
This work was supported National Institutes of Health Grant GM-52382 (L.S.B.).

Manuscript received January 17, 2006 and in revised form February 9, 2006.

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