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

Abstract

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 gamma 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.

Full text

thematic review
Thematic review series: Lipid Posttranslational Modifications
Structural biology of protein farnesyltransferase and
geranylgeranyltransferase type I
Kimberly T. Lane and Lorena S. Beese
1
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710
Abstract More than 100 proteins necessary for eukaryotic
cell growth, differentiation, and morphology require post-
translational 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 g 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 prenyl-
transferases) to establish a framework for understanding
the molecular basis of substrate specificity and mechanism.
These enzymes have been identified in a number of spe-
cies, including mammals, fungi, plants, and protists.
Prenyltransferase structures include complexes that rep-
resent the major steps along the reaction path, as well as
a num ber of complexes with clinically releva nt inhibi-
tors.
Such complexes may assist in the design of inhibi-
tors that could lead to treatments for cancer, viral infection,
and a number of deadly parasitic diseases.—Lane, K. T., and
L. S. Beese. Structural biology of protein farnesyltransfer-
ase and geranylgeranyltransferase type I. J. Lipid Res. 2006.
47: 681–699.
Supplementary key words prenyltransferase
.
isoprenoid
.
Ras
.
G
protein
.
cancer target
.
drug design
.
farnesyltransferase inhibitor
.
crystal structure
More than 100 proteins necessary for eukaryotic cell
growth, differentiation, and morphology require post-
translational modification by the covalent attachment of
an isoprenoid lipid (prenylation) (1). This modification is
catalyzed by three protein prenyltransferases: protein
farnesyltransferase (FTase) and protein geranylgeranyl-
transferase type I (GGTase-I), collectively termed the CaaX
prenyltransferases, as well as protein GGTase-II (or
RabGGTase) [reviewed in this series in (2)], whose sub-
strates 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 bio-
chemical properties of these enzymes have been reviewed
extensively elsewhere (1, 10–15).
After covalent attachment of the isoprenoid in the cyto-
plasm, most CaaX proteins undergo two further prenyla-
tion-dependent processing steps at the endoplasmic
reticulum (1): proteolytic removal of the aaX tripeptide
by the CaaX protease Ras and a-factor-converting enzyme
(Rce1), and carboxymethylation of the prenylcysteine
residue by the enzyme Isoprenylcysteine carboxyl methyl-
transferase (Icmt). The fully processed proteins exhibit
high affinity for cellular membranes and present a unique
structure at their C termini that can serve as a specific
recognition motif in certain protein-protein interactions
(Fig. 2).
FTase and GGTase-I were first identified in 1990 and
1991, respectively (3, 16). Both enzymes have been iden-
tified in a number of species, including mammals (17–21),
fungi (22–25), plants (26, 27), and protists (28, 29); the
genes encoding FTase and GGTase-I have been cloned
from several of these species (20, 30–35). These enzymes
have been shown to be essential for the function of these
organisms: elimination of these enzymes results in severe
defects or lethality in many instances (22, 26, 36–38).
Manuscript received 17 January 2006 and in revised form 9 February 2006.
Published, JLR Papers in Press, February 13, 2006.
DOI 10.1194/jlr.R600002-JLR200
Abbreviations: FPP, farnesyl diphosphate; FTase, protein farnesyl-
transferase; FTI, farnesyltransferase inhibitor; GGPP, geranylgeranyl
diphosphate; GGTase-I, geranylgeranyltransferase type I; GTI, geranyl-
geranyltransferase inhibitor; PDB ID, Protein Data Bank identifier.
1
To whom correspondence should be addressed.
e-mail: lsb@biochem.duke.edu
Copyright D 2006 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 47, 2006 681

Since the initial discovery of farnesylated fungal pro-
teins, 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 g subunit of heterotrimeric G proteins (53, 54), cen-
tromeric 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 trans-
duction (53, 62–64). Defects in these proteins are asso-
ciated with a number of human diseases, including Batten
disease (65), autosomal recessive retinitis pigmentosa and
autosomal dominant retinitis pigmentosa (66–68), Cana-
van 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 z20–30% of all human
tumors, making the CaaX prenyltransferases targets for
inhibition in cancer therapeutics (73, 74) (see below).
STRUCTURAL CHARACTERIZATION OF THE
CaaX PRENYLTRANSFERASES
Over the past decade, high-resolution crystal structures
of FTase and GGTase-I complexed with various substrates,
products, and inhibitors have advanced our understand-
ing of these enzymes. Structures representing each of the
major steps along the reaction pathway of CaaX prenyl-
transferases 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, fol-
lowed by the CaaX substrate (77–80). The release of
farnesylated product is the rate-limiting step of the re-
action 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. Al-
though the GGTase-I reaction has not been studied in
such detail, available evidence indicates that it proceeds
through a similar pathway (83, 84).
Overall enzyme structure
FTase and GGTase-I exist as ab heterodimers and share
a common a subunit (48 kDa molecular mass) (16, 30)
and a homologous b subunit (46 kDa in FTase, 42 kDa in
GGTase-I) (3, 19, 21, 31). Both a and b subunits are com-
posed primarily of a helices, which are arranged in a
crescent-shaped superhelix (a subunit) wrapped around
an a-a barrel (b subunit) (Fig. 4A, B) (76, 85).
The structure of the common a subunit is almost
identical in both FTase and GGTase-I, with only slight
shifts attributable to differences in the interactions with
the divergent b subunit. The helices of the a subunit are
folded into seven successive pairs, in a series of right-
handed antiparallel coiled coils. These “helical hairpins”
are arranged in a crescent-shaped superhelix that wraps
around the b subunit. The N-terminal domain (z50
Fig. 1. Biochemistry of the CaaX prenyltransferases. Ala, alanine; Gln, glutamine; Leu, leucine; Met,
methionine; Phe, phenylalanine; Ser, serine.
682 Journal of Lipid Research Volume 47, 2006

amino acids) is disordered in all complexes of FTase and
GGTase-I. It has been suggested that this proline-rich
domain may interact with other cellular factors, perhaps
functioning in enzyme localization, as deletion of this do-
main does not affect the catalytic activity (86) or the struc-
ture of the rest of the protein (87). In the absence of such
partners, this domain may be unfolded.
Although the b subunits of FTase and GGTase-I share
only 25% sequence identity, they have very similar
structures, consisting of 14 a helices in FTase and 13 in
GGTase-I. Twelve of the a helices are folded into an a-a
barrel (Fig. 4C). Six parallel helices (3b,5b,7b,9b,11b,
and 13b) create the core of the barrel. The other six
helices (2b,4b,6b,8b,10b, and 12b) are parallel with
one another and antiparallel with respect to the core he-
lices 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 b 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 A
˚
and a
depth of 14 A
˚
. 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 a and b subunits form an extensive interface,
burying .3,000 A
˚
2
,orz20%, of accessible surface area of
each subunit (76, 85). The a/b 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.
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.
Structural biology of protein FTase and GGTase-I 683

Zinc binding site
Both FTase and GGTase-I are zinc metalloenzymes,
binding one Zn
21
ion per protein dimer (89, 90). Crystal
structures of FTase and GGTase-I clearly indicate the
presence of a single Zn
21
ion bound to the b subunit near
the subunit interface (76, 85). In both enzyme structures,
the Zn
21
ion is coordinated by three strictly conserved res-
idues: D297b,C299b, and H362b in FTase and D269b,
C271b,andH321b in GGTase-I. Mutagenesis studies per-
formed on FTase are consistent with Zn
21
coordination by
residues D297b,C299b,andH362b (91–93) and addition-
ally implicate D359b in the binding of zinc (92). Crystal
structures of FTase (85, 87) reveal a stabilizing hydro-
gen bond between D359b and the zinc ligand H362b,
consistent with a secondary coordination shell effect ex-
erted 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 Zn
21
ion is required for catalytic activ-
ity and that it coordinates the cysteine thiol of the CaaX
substrate (12, 19, 32, 81, 89). Comparison of zinc coordina-
tion from high-resolution substrate [1.8 A
˚
(97)] and prod-
uct [1.65 A
˚
(K. L. Terry et al., unpublished observations)]
complexes indicates that the CaaX cysteine thiol(ate/ether)
forms a short (2.3 A
˚
) interaction with the zinc ion in sub-
strate complexes and a slighter longer (2.6 A
˚
) interaction in
the product complex (Fig. 5). The complete zinc coordina-
tion sphere for the substrate complex has four close inter-
actions (D297b,2.0A
˚
;C299b, 2.3 A
˚
;H362b,2.1A
˚
;CaaX
cysteine, 2.3 A
˚
) and one longer interaction (D297b,2.6A
˚
;
making this residue a bidentate ligand). The prenylated
peptide product complex has a similar coordination sphere
(D297b, 2.0 and 2.4 A
˚
;C299b,2.3A
˚
;H362b, 2.2 A
˚
;CaaX
prenyl cysteine, 2.6 A
˚
). 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
D297b, in substrate complexes appear to be sufficiently
weak to not be detected by this technique.
Isoprenoid diphosphate binding in binary complexes
In the cocrystal structures of FTase and GGTase-I with
FPP and GGPP prenyl substrate, respectively [Protein Data
Bank identifiers (PDB IDs) 1FT2 and 1N4P], the iso-
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 a subunit shown in red and the b 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).
684 Journal of Lipid Research Volume 47, 2006

prenoid binds along one side of the hydrophobic cavity of
the b subunit a-a barrel (Fig. 6A, B; complex 1 in Fig. 3)
(76, 99). The diphosphate moiety of both FPP and GGPP
binds in a positively charged cleft at the rim of the a-a
barrel near the a/b subunit interface. This group forms
several hydrogen bonds in each enzyme (K164a, H248b,
R291b, and Y300b in FTase; K164a, H219b, R263b, K266b,
and Y272b in GGTase-I). The farnesyl portion of FPP
binds in an extended conformation along one side of
the hydrophobic cavity of the a-a barrel, interacting with
a number of conserved aromatic residues. The first three
isoprene units of GGPP bind in a similar conformation
(with similar hydrophobic interactions with aromatic
residues in the GGTase-I active site), but the fourth iso-
prene unit is turned z 908 relative to the rest of the mol-
ecule. This conformation is significantly different from
that of the other known structures with bound isopren-
oids [i.e., FTase (99), RhoGDP dissociation inhibitor
(GDI) (47), and the phosducin-G
t
bg complex (100)], in
which the isoprenoid is completely extended. Superposi-
tion of GGTase-I and RabGGTase (101) by alignment of
all enzyme C
a
atoms illustrates the similarity of their
respective GGPP binding sites; the 24 residues surround-
ing the GGPP molecule in GGTase-I are identical or struc-
turally conservative substitutions in RabGGTase. The
high degree of homology in the prenyl binding site in-
dicates a similar mode of binding for the GGPP molecule
in RabGGTase.
Isoprenoid diphosphate substrate specificity:
the ruler hypothesis
CaaX prenyltransferase activities are highly selective for
their isoprenoid diphosphate substrates: FTase for FPP
and GGTase-I for GGPP. Binding studies demonstrate,
Fig. 4. Structures of the CaaX prenyltransferases. Overall structures of FTase (A) and geranylgeranyl-
transferase type I (GGTase-I) (B), with the a subunit shown in red, the b 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 b subunit of these enzymes.
Structural biology of protein FTase and GGTase-I 685

however, that FTase can bind both FPP and GGPP with
nanomolar affinity, yet the enzyme is unable to transfer
the geranylgeranyl group to substrate proteins (17, 78, 79,
89, 102), indicating that GGPP is an inhibitor of FTase.
GGTase-I similarly binds both FPP and GGPP, although it
binds FPP with somewhat lower affinity (79, 103). Unlike
FTase, GGTase-I can (mis)transfer a farnesyl group to
substrate proteins, but with reduced efficiency (83).
Superposition of FTase and GGTase-I in binary isopren-
oid substrate complexes by aligning all enzyme C
a
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 indistinguish-
able. 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 (W102b and Y365b),
whereas the corresponding positions in GGTase-I (and
RabGGTase) are occupied by the smaller residues threo-
nine and phenylalanine to accommodate the fourth iso-
prene unit of the GGPP molecule, which cannot fit in
FTase. Therefore, FTase and GGTase-I can function as
simple length-discriminating molecular rulers. This hypoth-
esis was tested by introducing mutations into FTase that
are predicted to convert it to a geranylgeranyltransferase
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
(D297b, C299b, and H362b) as well as the cysteine (Cys) of the CaaX motif. Note the longer interaction
distance between the Zn
21
ion and the CaaX cysteine in the product complex.
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).
686 Journal of Lipid Research Volume 47, 2006

(76). A mutation at the W102b residue of FTase to a
threonine(thecorresponding 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).
Peptide and isoprenoid binding in ternary complexes
Crystal structures of the CaaX prenyltransferases with
bound peptide substrates and nonreactive isoprenoid
analogs revealed the conformation of the bound CaaX
substrate (complex 2 in Fig. 3) (76, 94, 95). Each of these
FTase and GGTase-I complexes includes a peptide derived
from the K-Ras4B (CVIM Ca
1
a
2
X sequence; PDB IDs 1D8D
and 1QBQ) or Rap2b (CVIL Ca
1
a
2
X sequence; PDB ID
1N4Q) protein substrate, respectively, taking advantage of
the ability of these enzymes to prenylate peptides as short
as the CaaX tetrapeptide (3, 6, 18). The structure of the
enzyme in these complexes is essentially identical to the
previously determined structures, with the exception of a
few minor side chain rearrangements in the vicinity of the
diphosphate binding site compared with the unliganded
FTase structure (PDB ID 1FT1) (85).
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, par-
ticularly the a
2
and X residues (Fig. 8A, B). The peptide
substrate sandwiches the isoprenoid against the wall of the
hydrophobic cavity, burying 115 and 140 A
˚
2
(in FTase and
GGTase-I, respectively) of accessible isoprenoid diphos-
phate 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 diphos-
phate substrate molecule were also observed to interact
with the analogs. These structures, therefore, are consis-
tent with the ordered binding pattern observed by kinetics
in which isoprenoid diphosphate binding precedes
peptide binding (77, 78).
In the FTase and GGTase-I ternary substrate complexes,
the peptide Ca
1
a
2
X motif binds in an extended confor-
mation in the hydrophobic cavity of the b subunit. As
predicted from spectroscopic studies (81), the cysteine
residue of the peptide Ca
1
a
2
X sequence directly coordi-
nates the zinc ion (Fig. 5). The side chain of the peptide
valine residue (position a
1
) points into solvent without
making any hydrophobic interactions with the isoprenoid.
The isoleucine side chain of the peptide substrate (a
2
)is
positioned in close proximity to the isoprenoid, making a
hydrophobic interaction, whereas the backbone carbonyl
oxygen of this residue participates in a hydrogen bond
with the enzyme (R202b in FTase, R173b in GGTase-I).
The C-terminal carboxylate of the X residue (methionine
in FTase, leucine in GGTase-I) forms hydrogen bonds with
Q167a and a buried water molecule coordinated by three
enzyme residues (H149b, E198b, and R202b in FTase;
H121b, E169b, and R173b in GGTase-I). The residue in
position X is also in van der Waals contact with a number
of enzyme residues (Y131a, A98b, S99b, W102b, H149b,
A151b, and P152b in FTase; T49b, H121b, A123b, and
F174b in GGTase-I). These interactions bury a large por-
tion of the accessible surface area of the residue in position
X. As noted, the only directional, bonded interactions
between the peptide CaaX sequence and the protein are
the Zn
21
-to-cysteine thiol(ate) interaction and the hydro-
gen bonds involving the carbonyl oxygen of the a
2
iso-
leucine residue and the C-terminal carboxylate. Together,
these interactions help anchor the CaaX peptide into its
observed substrate binding conformation.
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 1 Ras-CVLS (blue), FPP 1 Ras-CVLL (green), GGPP 1 Ras-CVLS (yellow), and GGPP 1 Ras-CVLL (red).
Reproduced with permission from (76).
Structural biology of protein FTase and GGTase-I 687

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 struc-
tures, proving that zinc is not required to maintain the
proper three-dimensional fold of FTase. Furthermore, the
conformations of the zinc ligands (D297b, C299b, and
H362b) 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 a
2
and X residues of the CaaX peptide bind in the
same manner as in the presence of zinc, whereas the cys-
teine and valine residues bind much differently, forming a
b-turn. The cysteine thiol(ate) has shifted z9A
˚
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 pep-
tide 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 Ca
1
a
2
X 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 pre-
nyltransferases, structures were determined of FTase and
GGTase-I complexed with the appropriate isoprenoid di-
phosphate 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 g
2
subunit (CAIL; PDB ID 1TNY in GGTase-I) (97).
A superposition of these ternary complexes by aligning
all C
a
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 an-
chor points within the recognition motif (cysteine and
C terminus) that make specific interactions with the en-
zyme 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 a
1
residue is solvent-exposed and
can accommodate any amino acid, as observed in solution
studies (6, 76, 94, 95). Both the a
2
and X positions are buried
in the active site; consequently, they are the major deter-
minants of peptide selectivity through steric and electro-
static complementarity between the amino acid side chains
and enzyme residues.
In these structural studies, it was observed that the
binding sites on FTase and GGTase-I that recognize the a
2
peptide residue have unique steric and aromatic proper-
ties, suggesting that the a
2
residue may affect Ca
1
a
2
X
recognition. The structures indicate that the a
2
binding
site can accommodate a variety of amino acids, including
valine, isoleucine, leucine, phenylalanine, tyrosine, pro-
line, threonine, and methionine, consistent with previous
studies of FTase peptide selectivity in mammalian and
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).
688 Journal of Lipid Research Volume 47, 2006

yeast systems (6, 104). Comparison of these sites illustrates
the increased aromatic character of FTase relative to
GGTase-I, which has subtle effects on CaaX specificity but
a more marked effect on inhibitor binding (see below)
(Fig. 10).
The X position is the primary sequence determinant
that specifies whether a peptide is a substrate for FTase,
GGTase-I, both, or neither. The structures reveal that
FTase and GGTase-I have different X-residue binding
pockets, each with distinct electrostatic properties. The X-
residue binding pocket in FTase is more polar and accepts
the residues methionine, serine, and glutamine (as well as
alanine, threonine, and cysteine to a lesser extent). This
pocket in GGTase-I has more hydrophobic character and
accepts leucine and phenylalanine (as well as isoleucine
and valine to a lesser extent). Both pockets discriminate
against bulky amino acids such as tyrosine, tryptophan,
and arginine through steric complementarity. Interest-
ingly, this study also revealed a previously unobserved
secondary X residue binding site in FTase, in which the
C-terminal phenylalanine residue of the TC21-derived
peptide binds. This pocket may also accommodate leucine,
asparagine, or histidine.
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 prenyl-
transferase substrates not identified previously.
Product formation
In both FTase and GGTase-I, the structures of the prod-
uct complex (complex 3 in Fig. 3) (PDB IDs 1KZP and
1N4R) and ternary substrate complexes are virtually iden-
tical (75). There are no observed changes in the poly-
peptide 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 pyro-
phosphate product release. Equally, the peptide exhibits
no appreciable differences between its reactant and prod-
uct structures (Fig. 11A). The isoprenoid, however, moves
toward the peptide in the product structure, with the C
1
atom nearly 7 A
˚
closer to the Zn
21
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
Fig. 9. Comparison of Ca
1
a
2
X 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 resi-
dues. Reproduced with permission from (97).
Fig. 10. Comparison of the a
2
binding site in FTase and GGTase-I.
Superposition of FTase and GGTase-I shows residues that interact
with the a
2
residue of the Ca
1
a
2
X motif (PDB IDs 1D81 and 1N4Q).
Regions of the FTase ternary complex forming the a
2
binding site
(enzyme residues W102b, W106b, and Y361b and isoprene 3) are
colored red. Corresponding regions of the GGTase-I ternary
complex (enzyme residues T49b, F53b, and L321b, 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 a
2
site are also shown.
Reproduced with permission from (97).
Structural biology of protein FTase and GGTase-I 689

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.
Product release
We note that the product complex described above
(complex 3 in Fig. 3) does not represent the final step in
this reaction. As described above, the rate-limiting step of
the prenylation reaction is product release (78, 105),
requiring binding of the next substrate(s) (82). This final
step is represented by structures of FTase and GGTase-I
complexed with both isoprenoid substrate and prenylated
peptide product (complex 4 in Fig. 3) (PDB IDs 1KZO and
1N4S), which reveal that binding of the fresh isoprenoid
diphosphate substrate molecule moves the prenyl group
of the prenylated peptide to a new binding site, the “exit
groove” (Fig. 11B) (75, 76). This movement is accompa-
nied by a conformational change in the CaaX moiety.
Therefore, there are two CaaX peptide conformations in
the product: extended (complex 3 in Fig. 3) and type I
b-turn (complex 4 in Fig. 3). NMR studies of peptides
bound to FTase revealed a similar b-turn, which was
postulated to correspond to a reactant rather than a
product complex (106, 107). In the displaced product, the
X and a
2
residues remain in their original positions,
indicating that the peptide substrate is competitive with
respect to the displaced product. The three C-terminal
amino acids of the prenylated peptide product make
extensive van der Waals contacts with the new isoprenoid
substrate, suggesting that the amino acid sequence of the
CaaX motif may modulate product release. Multiple
binding sites for product molecules have been observed
in processive enzyme systems, in which substrate binding
is involved in product release or translocation [e.g., the P
and E sites of the ribosome (108)]. We note that this
complex represents a direct observation of both substrate
and product bound simultaneously to an enzyme that
catalyzes an apparently nonprocessive reaction.
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 disso-
ciation of the monoprenylated intermediate (109) [re-
viewed 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 A
˚
diameter hydrophobic tunnel, originating
from a shallow groove analogous to the exit groove in
FTase and GGTase-I and extending through the b 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 ex-
plain 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,
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-b turn, placing the farnesyl portion
(brown) of the product in a conserved “exit groove” (residues
shown in cyan). Reproduced with permission from (75).
690 Journal of Lipid Research Volume 47, 2006

it is shielded from the cytoplasm, preventing aggrega-
tion or association with an incorrect membrane compart-
ment. 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 devel-
oped a transition state model for catalysis by CaaX prenyl-
transferases consistent with the observed structural and
kinetic information (Fig. 12) (75, 76). The proposed
model retains elements of both the postulated electro-
philic and nucleophilic components of the reaction mech-
anism (80, 113, 114). In this model, the CaaX motif binds
as observed in the ternary substrate complex, coordinating
the Zn
21
ion, whereas the isoprenoid diphosphate rotates
through its second isoprene unit to bring the C
1
atom into
alignment for thioether bond formation, maintaining the
binding conformation of the b-phosphate in its substrate
conformation. In this reaction, the isoprenoid C
1
atom
undergoes inversion of configuration during the reaction
(115–117). The developing negative charge on the di-
phosphate moiety (localized mainly on the a-phosphate) is
stabilized by several interactions with the enzyme, partic-
ularly residues K164a and a b subunit tyrosine (Y300b in
FTase, Y272b in GGTase-I). Kinetic studies demonstrated
that FTase, but not GGTase-I, requires millimolar levels of
Mg
21
for full catalytic efficiency (19, 89). It has been hy-
pothesized that the Mg
21
ion required by FTase stabilizes
the developing negative charge on the diphosphate as the
bond breaks between the a-phosphate and the C
1
atom of
the farnesyl group. Structural studies suggest that Mg
21
is
coordinated by residue D352b of the enzyme and the FPP
diphosphate moiety (75). Sequence alignments reveal that
in GGTase-I, a lysine occupies the position corresponding
to D352b 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
e
at the site of Mg
21
in FTase,
allowing it to stabilize the diphosphate group. Mutagenesis
studies (118, 119) indicate that Mg
21
sensitivity is depen-
dent on these two residues; a lysine at 352b of FTase abol-
ishes Mg
21
dependence, whereas an aspartate at 311b in
GGTase-I introduces Mg
21
dependence.
INHIBITION OF CaaX
PRENYLTRANSFERASE ACTIVITY
Three major classes of farnesyltransferase inhibitors
(FTIs) and geranylgeranyltransferase inhibitors (GTIs)
have been identified: isoprenoid diphosphate-derived,
peptide-competitive (peptidomimetic and nonpeptidomi-
metic), 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 anti-
cancer (121–125), antiparasitic (29, 126–128), antifungal
(129–131), and antiviral (132–134) therapeutic agents.
Some of these have been characterized by X-ray crystallo-
graphic studies, revealing a molecular basis for their inhib-
itory activities.
Isoprenoid diphosphate-derived inhibitors
A number of FPP and GGPP analogs have been de-
veloped 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 hydro-
phobic farnesyl group and a negatively charged moiety
mimicking the diphosphate (138), consistent with the in-
teractions 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
Fig. 12. Proposed transition state model for the CaaX prenyl-
transferases. The scissile phosphoether bond between the diphos-
phate 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 D352b, in FTase is replaced by K311b in
GGTase-I. Lys, lysine.
Structural biology of protein FTase and GGTase-I 691

activity, because elongation of the farnesyl group by a
single carbon resulted in a decrease in IC
50
of .200-fold,
consistent with the structure-derived ruler hypothesis.
Several isoprenoid analog inhibitors have been charac-
terized 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 a-hydroxyfar-
nesylphosphonic acid (94) and the GGPP analog 39 -aza
GGPP (76, 97). In each of these structures, the isoprenoid
analogs bind in a manner similar to their substrate coun-
terparts, interacting extensively with both the enzyme and
the CaaX substrate. These inhibitors continue to be invalu-
able 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 dem-
onstrated that this enzyme is inhibited by short Ca
1
a
2
X
peptides (3, 4, 6). The most effective inhibitory peptides
contain a nonpolar aliphatic or aromatic amino acid in the
a
1
or a
2
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 a
2
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
Fig. 13. Comparison of CaaX nonsubstrate and substrate conformations. A: CaaX tetrapeptide nonsub-
strate 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 non-
substrate tetrapeptide inhibitor CVFM. A boxed region highlights the differences between the substrate
and nonsubstrate conformations. D: Energetically unfavorable steric contact (red spikes) between the C
g2
methyl group of the isoleucine residue in the a
2
position and the carbonyl oxygen of the a
1
residue when the
phenylalanine of the CVFM tetrapeptide is replaced with isoleucine. Consequently, a tetrapeptide with a
b-branched residue (isoleucine or valine) in the a
2
position cannot adopt the nonsubstrate binding mode
observed for CVFM. Reproduced with permission from (96).
692 Journal of Lipid Research Volume 47, 2006

catalytic activity (4, 7). Because these molecules are pep-
tides, they have three significant disadvantages for poten-
tial in vivo activity: poor cellular uptake, rapid degradation,
and FTase farnesylation (in some cases), which results in
depletion of the inhibitor. To overcome these disadvan-
tages, a number of peptidomimetic inhibitors have been
developed and characterized (96, 121, 146–150).
The mechanism for tetrapeptide inhibition was stud-
ied 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), de-
veloped by Merck Research Laboratories (96). This inhibi-
tor compound, mimicking the tetrapeptide CIFM, is the
metabolic product of the prodrug L-744,832; administra-
tion 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 inhibi-
tors (the nonsubstrate CVFM peptide and L-739,750)
adopt a similar conformation, in which there is an z3A
˚
distance shift of the inhibitor cysteine C
a
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 a-phosphate of the
FPP molecule, as well as by hydrophobic interactions be-
tween the phenylalanine-derived side chain of the inhibi-
tor 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 move-
ment 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 a
2
position adopts essentially the same conformation;
this conformation places steric constraints on the f back-
bone angle between the a
1
and a
2
residues. b-Branched
amino acids (isoleucine and valine) in the a
2
position form
an unfavorable steric interaction between their C
g2
methyl
group and the carbonyl oxygen of the a
1
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 a
2
position are substrates, whereas peptides with other non-
branched aliphatic amino acids are poor substrates or non-
substrates (provided that the N terminus is not blocked).
Nonpeptide inhibitors. Another class of peptide-competi-
tive inhibitors includes molecules that are not derived
from peptides but still bind in the CaaX binding site of the
enzyme; this class is by far the most prevalent and the most
characterized (122–125, 144, 151–165). This class of in-
hibitors includes compounds produced by the drug dis-
covery and design efforts of Merck Research Laboratories,
Janssen Research Foundation, Bristol-Myers Squibb Co.,
Schering-Plough, and Abbott Laboratories, some of which
have been evaluated in phase I, II, and III clinical trials for
the treatment of a variety of human cancers (122, 123).
Crystal structures of a number of these inhibitors com-
plexed with the CaaX prenyltransferases have been
determined. In each of these structures, the compounds
bind in the peptide binding site, typically without inducing
large structural changes in the enzyme. Each of these
compounds participates in extensive hydrophobic inter-
actions with both the enzyme and the FPP/FPP analog
Fig. 14. FTase inhibitors R115777, BMS-214662, and ABT-839.
Ribbon cartoons of FTase complexed with FPP or FPP analog
a-hydroxyfarnesylphosphonic acid (a-HFP)(purple) and the in-
hibitor 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 Zn
21
ion (magenta), coordinated by the imidizole
group of R115777 and BMS-214662.
Structural biology of protein FTase and GGTase-I 693

molecule, as well as in a combination of direct and water-
mediated hydrogen bonds with the enzyme.
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 inhibi-
tors bind in the FTase peptide binding site, overlapping
with the binding of the Ca
1
a
2
portion of the substrate
Ca
1
a
2
X peptide (Fig. 14). R11577 and BMS-214662 coor-
dinate the catalytic Zn
21
ion via an imidazole group; how-
ever, 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 ex-
tends into the X residue binding site.
Design efforts from Schering-Plough have resulted in a
number of FTase inhibitors, of which a series of tricyclic
compounds have been structurally characterized (144).
These compounds are composed of three connected rings,
with a tail containing two or more rings extending from the
central seven carbon ring, representing a series of mono-,
di-, and trihalogenated species. SCH66336, an inhibitor in
this series, is observed in an unusual conformation in the
FTase active site (PDB ID 1O5M) (Fig. 15). This compound
does not coordinate the Zn
21
ion and only overlaps the
a
1
and a
2
portion of the substrate Ca
1
a
2
X peptide. Addi-
tionally, a large portion of the inhibitor is observed binding
in the exit groove described above, where the displaced
prenyl group of the farnesylated peptide product binds in
the translocated product complex. The latter observation
may explain the binding affinity toward FTase that this
inhibitor exhibits, despite its lack of Zn
21
coordination.
Although most of these inhibitors are highly selective
toward FTase in preference to GGTase-I, L-778,123 (Merck
Research Laboratories) is a nonpeptide inhibitor of both
FTase and GGTase-I (154, 155). No steric blocks prevent
compounds such as R11577 and BMS-214662 from binding
in GGTase-I, although binding is not favored because the
GGTase-I a
2
binding site has a reduced aromatic character
that does not permit the stabilizing aromatic stacking in-
teractions (166, 167) observed in FTase (see above). These
differences in the a
2
binding site may cause the alternative
binding mode adopted by L-778,123 in GGTase-I (Fig. 16)
(143). In FTase-I (PDB ID 1S63), the peptide-competitive
binding mode is stabilized by aromatic stacking inter-
actions with the aromatic residues that constitute the a
2
binding site. In GGTase-I (PDB ID 1S64), these stacking
interactions are not possible, and L-778,123 adopts a
GGPP-competitive binding mode that permits the forma-
tion of a hydrogen bond in lieu of stacking interactions.
In this structure, the inhibitor also interacts with a bound
sulfate anion; this synergistic interaction may additionally
induce a small change in the GGPP binding site that en-
hances L-778,123 binding (168, 169).
Bisubstrate mimics
The final class of prenyltransferase inhibitors includes a
variety of molecules that mimic a bisubstrate complex and
are competitive for both the isoprenoid diphosphate and
CaaX binding sites. Several of these inhibitors have been
synthesized and characterized (170–172), although none
has been studied by structural methods.
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 a
2
residue of the
Ca
1
a
2
X peptide, the “a
2
binding site,” and a conserved arginine
(Arg) residue are shown. Aromatic stacking interactions are not
possible between L-778,123 and the GGTase-I a
2
binding site, en-
couraging the inhibitor to adopt a lipid-competitive binding mode
that permits the formation of a hydrogen bond. Reproduced with
permission from (143).
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 pep-
tide CVIM (dark gray) (PDB ID 1D8D) and (B) the par-
tially displaced peptide product (light gray) (PDB ID
1KZO). Also shown is the Zn
2+
ion (magenta).
694 Journal of Lipid Research Volume 47, 2006

STRUCTURAL ANALYSIS OF CaaX
PRENYLTRANSFERASE AND
HUMAN THERAPEUTICS
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 micro-
organisms, 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 im-
munocompromised 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 devel-
opment bypasses many of the hurdles involved in designing
drugs de novo (177). Structural studies of the protein pre-
nyltransferases from human pathogens would facilitate the
design of potent, highly selective inhibitors.
This work was supported National Institutes of Health Grant
GM-52382 (L.S.B.).
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