Refinement and prediction of protein prenylation motifs

We refined the motifs for carboxy-terminal protein prenylation by analysis of known substrates for farnesyltransferase (FT), geranylgeranyltransferase I (GGT1) and geranylgeranyltransferase II (GGT2). In addition to the CaaX box for the first two enzymes, we identify a preceding linker region that appears constrained in physicochemical properties, requiring small or flexible, preferably hydrophilic, amino acids. Predictors were constructed on the basis of sequence and physical property profiles, including interpositional correlations, and are available as the Prenylation Prediction Suite (PrePS, ) which also allows evaluation of evolutionary motif conservation. PrePS can predict partially overlapping substrate specificities, which is of medical importance in the case of understanding cellular action of FT inhibitors as anticancer and anti-parasite agents.     

Protein farnesyltransferase and protein prenylation in Plasmodium falciparum

Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.     

Isoprenoids and Related Pharmacological Interventions: Potential Application in Alzheimer’s Disease

Two major isoprenoids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, serve as lipid donors for the posttranslational modification (known as prenylation) of proteins that possess a characteristic C-terminal motif. The prenylation reaction is catalyzed by prenyltransferases. The lipid prenyl group facilitates to anchor the proteins in cell membranes and mediates protein-protein interactions. A variety of important intracellular proteins undergo prenylation, including almost all members of small GTPase superfamilies as well as heterotrimeric G protein subunits and nuclear lamins. These prenylated proteins are involved in regulating a wide range of cellular processes and functions, such as cell growth, differentiation, cytoskeletal organization, and vesicle trafficking. Prenylated proteins are also implicated in the pathogenesis of different types of diseases. Consequently, isoprenoids and/or prenyltransferases have emerged as attractive therapeutic targets for combating various disorders. This review attempts to summarize the pharmacological agents currently available or under development that control isoprenoid availability and/or the process of prenylation, mainly focusing on statins, bisphosphonates, and prenyltransferase inhibitors. Whereas statins and bisphosphonates deplete the production of isoprenoids by inhibiting the activity of upstream enzymes, prenyltransferase inhibitors directly block the prenylation of proteins. As the importance of isoprenoids and prenylated proteins in health and disease continues to emerge, the therapeutic potential of these pharmacological agents has expanded across multiple disciplines. This review mainly discusses their potential application in Alzheimer's disease.     

Isoprenoids and protein prenylation: implications in the pathogenesis and therapeutic intervention of Alzheimer’s disease

The mevalonate–isoprenoid–cholesterol biosynthesis pathway plays a key role in human health and disease. The importance of this pathway is underscored by the discovery that two major isoprenoids, farnesyl and geranylgeranyl pyrophosphate, are required to modify an array of proteins through a process known as protein prenylation, catalyzed by prenyltransferases. The lipophilic prenyl group facilitates the anchoring of proteins in cell membranes, mediating protein–protein interactions and signal transduction. Numerous essential intracellular proteins undergo prenylation, including most members of the small GTPase superfamily as well as heterotrimeric G proteins and nuclear lamins, and are involved in regulating a plethora of cellular processes and functions. Dysregulation of isoprenoids and protein prenylation is implicated in various disorders, including cardiovascular and cerebrovascular diseases, cancers, bone diseases, infectious diseases, progeria, and neurodegenerative diseases including Alzheimer’s disease (AD). Therefore, isoprenoids and/or prenyltransferases have emerged as attractive targets for developing therapeutic agents. Here, we provide a general overview of isoprenoid synthesis, the process of protein prenylation and the complexity of prenylated proteins, and pharmacological agents that regulate isoprenoids and protein prenylation. Recent findings that connect isoprenoids/protein prenylation with AD are summarized and potential applications of new prenylomic technologies for uncovering the role of prenylated proteins in the pathogenesis of AD are discussed.     

Protein prenyltransferases

Three different protein prenyltransferases (farnesyltransferase and geranylgeranyltransferases I and II) catalyze the attachment of prenyl lipid anchors 15 or 20 carbons long to the carboxyl termini of a variety of eukaryotic proteins. Farnesyltransferase and geranylgeranyltransferase I both recognize a 'Ca₁a₂X' motif on their protein substrates; geranylgeranyltransferase II recognizes a different, non-CaaX motif. Each enzyme has two subunits. The genes encoding CaaX protein prenyltransferases are considerably longer than those encoding non-CaaX subunits, as a result of longer introns. Alternative splice forms are predicted to occur, but the extent to which each splice form is translated and the functions of the different resulting isoforms remain to be established. Farnesyltransferase-inhibitor drugs have been developed as anti-cancer agents and may also be able to treat several other diseases. The effects of these inhibitors are complicated, however, by the overlapping substrate specificities of geranylgeranyltransferase I and farnesyltransferase.     

Synthetic isoprenoid analogues for the study of prenylated proteins: Fluorescent imaging and proteomic applications

Protein prenylation is a posttranslational modification catalyzed by prenyltransferases involving the attachment of farnesyl or geranylgeranyl groups to residues near the C-termini of proteins. This irreversible covalent modification is important for membrane localization and proper signal transduction. Here, the use of isoprenoid analogues for studying prenylated proteins is reviewed. First, experiments with analogues containing small fluorophores that are alternative substrates for prenyltransferases are described. Those analogues have been useful for quantifying binding affinity and for the production of fluorescently labeled proteins. Next, the use of analogues that incorporate biotin, bioorthogonal groups or antigenic moieties is described. Such probes have been particularly useful for identifying proteins that are naturally prenylated within mammalian cells. Overall, the use of isoprenoid analogues has contributed significantly to the understanding of protein prenlation.     

A cell-specific,prenylation-independent mechanism regulates targeting of type II RACs

The RHO proteins, which regulate numerous signaling cascades, undergo prenylation, facilitating their interaction with membranes and with proteins called RHO.GDP dissociation inhibitors. It has been suggested that prenylation is required for RHO function. Eleven RHO-related proteins were identified in Arabidopsis. Eight of them are putatively prenylated. We show that targeting of the remaining three proteins, AtRAC7, AtRAC8, and AtRAC10, is prenylation independent, requires palmitoylation, and occurs by a cell-specific mechanism. AtRAC8 and AtRAC10 could not be prenylated by either farnesyltransferase or geranylgeranyltransferase I, whereas AtRAC7 could be prenylated by both enzymes in yeast. The association of AtRAC7 with the plasma membrane in plants did not require farnesyltransferase or a functional CaaX box. Recombinant AtRAC8 was palmitoylated in vitro, and inhibition of protein palmitoylation relieved the association of all three proteins with the plasma membrane. Interestingly, AtRAC8 and a constitutively active mutant, Atrac7mV(15), were not associated with the plasma membrane in root hair cells, whose elongation requires the localization of prenylated RHOs in the plasma membrane at the cell tip. Moreover, Atrac7mV(15) did not induce root hair deformation, unlike its prenylated homologs. Thus, AtRAC7, AtRAC8, and AtRAC10 may represent a group of proteins that have evolved to fulfill unique functions.     

The alpha-subunit of protein prenyltransferases is a member of the tetratricopeptide repeat family.

Lipidation catalyzed by protein prenyltransferases is essential for the biological function of a number of eukaryotic proteins, many of which are involved in signal transduction and vesicular traffic regulation. Sequence similarity searches reveal that the alpha-subunit of protein prenyltransferases (PTalpha) is a member of the tetratricopeptide repeat (TPR) superfamily. This finding makes the three-dimensional structure of the rat protein farnesyltransferase the first structural model of a TPR protein interacting with its protein partner. Structural comparison of the two TPR domains in protein farnesyltransferase and protein phosphatase 5 indicates that variation in TPR consensus residues may affect protein binding specificity through altering the overall shape of the TPR superhelix. A general approach to evolutionary analysis of proteins with repetitive sequence motifs has been developed and applied to the protein prenyltransferases and other TPR proteins. The results suggest that all members in PTalpha family originated from a common multirepeat ancestor, while the common ancestor of PTalpha and other members of TPR superfamily is likely to be a single repeat protein.     

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a₂ position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca₁a₂X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a₂ and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.     

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.     

Structure of protein geranylgeranyltransferase-I from the human pathogen Candida albicans complexed with a lipid substrate

Protein geranylgeranyltransferase-I (GGTase-I) catalyzes the transfer of a 20-carbon isoprenoid lipid to the sulfur of a cysteine residue located near the C terminus of numerous cellular proteins, including members of the Rho superfamily of small GTPases and other essential signal transduction proteins. In humans, GGTase-I and the homologous protein farnesyltransferase (FTase) are targets of anticancer therapeutics because of the role small GTPases play in oncogenesis. Protein prenyltransferases are also essential for many fungal and protozoan pathogens that infect humans, and have therefore become important targets for treating infectious diseases. Candida albicans, a causative agent of systemic fungal infections in immunocompromised individuals, is one pathogen for which protein prenylation is essential for survival. Here we present the crystal structure of GGTase-I from C. albicans (CaGGTase-I) in complex with its cognate lipid substrate, geranylgeranylpyrophosphate. This structure provides a high-resolution picture of a non-mammalian protein prenyltransferase. There are significant variations between species in critical areas of the active site, including the isoprenoid-binding pocket, as well as the putative product exit groove. These differences indicate the regions where specific protein prenyltransferase inhibitors with antifungal activity can be designed.     

Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I

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.     

Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry

To evaluate the ability of an insect cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.     

A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release

Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substrates include oncogenic Ras proteins, which are implicated in up to 30% of all human cancers, making prenyltransferases a viable target for chemotherapeutic drugs. A coupled assay has been developed to measure the rate constant of diphosphate (PPi) dissociation during the prenyltransferase reaction under both single and multiple turnover conditions. In this assay, the PPi group produced in the prenyltransferase reaction is rapidly cleaved by inorganic pyrophosphatase to form phosphate (Pi), which is then bound by a coumarin-labeled phosphate binding protein from Escherichia coli, resulting in a fluorescence increase. The observed rate constant for PPi release is equal to the rate constant of prenylation of the peptide, as measured by other assays, so that this nonradioactive assay can be used to measure prenyltransferase activity under either single or multiple turnover conditions. This assay can be adapted for high-throughput screening for potential prenyltransferase substrates and inhibitors.     

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon-carbon-based and carbon-oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism.     

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.     

A novel protein geranylgeranyltransferase-I inhibitor with high potency, selectivity, and cellular activity

Inhibiting protein prenylation is an attractive means to modulate cellular processes controlled by a variety of signaling proteins, including oncogenic proteins such as Ras and Rho GTPases. The largest class of prenylated proteins contain a so-called CaaX motif at their carboxyl termini and are subject to a maturation process initiated by the attachment of an isoprenoid lipid by either protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I). Inhibitors of FTase, termed FTIs, have been the subject of intensive development in the past decade and have shown efficacy in clinical trials. Although GGTase-I inhibitors (GGTIs) have received less attention, accumulating evidence suggests GGTIs may augment therapies using FTIs and could be useful to treat a myriad of additional disease states. Here we describe the characterization of a selective, highly potent, and cell-active GGTase-I inhibitor, GGTI-DU40. Kinetic analysis revealed that inhibition by GGTI-DU40 is competitive with the protein substrate and uncompetitive with the isoprenoid substrate; the Ki for the inhibition is 0.8 nM. GGTI-DU40 is highly selective for GGTase-I both in vitro and in living cells. Studies indicate GGTI-DU40 blocks prenylation of a number of geranylgeranylated CaaX proteins. Treatment of MDA-MB-231 breast cancer cells with GGTI-DU40 inhibited thrombin-induced cell rounding via a process that involves inhibition of Rho proteins without significantly effecting parallel mobilization of calcium via Gbetagamma. These studies establish GGTI-DU40 as a prime tool for interrogating biologies associated with protein geranylgeranylation and define a novel structure for this emerging class of experimental therapeutics.     

Protein prenyltransferases: anchor size,pseudogenes and parasites

Lipid modification of eukaryotic proteins by protein prenyltransferases is required for critical signaling pathways, cell cycle progression, cytoskeleton remodeling, induction of apoptosis and vesicular trafficking. This review analyzes the influence of distinct states of sequential posttranslational processing that can be obtained after single or double prenylation, reversible palmitoylation, proteolytic cleavage of the C-terminus and possible reversible carboxymethylation. This series of modifications, as well as the exact length of the prenyl anchor, are determinants in protein-membrane and specific protein-protein interactions of protein prenyltransferase substrates. Furthermore, the occurrence and distribution of pseudogenes of protein prenyltransferase subunits are discussed. Besides being developed as anti-cancer agents, prenyltransferase inhibitors are effective against an increasing number of parasitic diseases. Extensive screens for protein prenyltransferases in genomic data of fungal and protozoan pathogens unveil a series of new pharmacologic targets for prenyltransferase inhibition, including the parasites Brugia malayi, Onchocerca volvulus, Aspergillus nidulans, Pneumocystis carinii, Entamoeba histolytica, Strongyloides stercoralis, Trichinella spiralis and Cryptosporidium parvum.     

Conversion of protein farnesyltransferase to a geranylgeranyltransferase

Posttranslational modifications are essential for the proper function of a number of proteins in the cell. One such modification, the covalent attachment of a single isoprenoid lipid (prenylation), is carried out by the CaaX prenyltransferases, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type-I (GGTase-I). Substrate proteins of these two enzymes are involved in a variety of cellular functions but are largely associated with signal transduction. These modified proteins include members of the Ras superfamily, heterotrimeric G-proteins, centromeric proteins, and a number of proteins involved in nuclear integrity. Although FTase and GGTase-I are highly homologous, they are quite selective for their substrates, particularly for their isoprenoid diphosphate substrates, FPP and GGPP, respectively. Here, we present both crystallographic and kinetic analyses of mutants designed to explore this isoprenoid specificity and demonstrate that this specificity is dependent upon two enzyme residues in the beta subunits of the enzymes, W102beta and Y365beta in FTase (T49beta and F324beta, respectively, in GGTase-I).     

Crystallographic analysis of CaaX prenyltransferases complexed with substrates defines rules of protein substrate selectivity

Post-translational modifications are essential for the proper function of many proteins in the cell. The attachment of an isoprenoid lipid (a process termed prenylation) by protein farnesyltransferase (FTase) or geranylgeranyltransferase type I (GGTase-I) is essential for the function of many signal transduction proteins involved in growth, differentiation, and oncogenesis. FTase and GGTase-I (also called the CaaX prenyltransferases) recognize protein substrates with a C-terminal tetrapeptide recognition motif called the Ca1a2X box. These enzymes possess distinct but overlapping protein substrate specificity that is determined primarily by the sequence identity of the Ca1a2X motif. To determine how the identity of the Ca1a2X motif residues and sequence upstream of this motif affect substrate binding, we have solved crystal structures of FTase and GGTase-I complexed with a total of eight cognate and cross-reactive substrate peptides, including those derived from the C termini of the oncoproteins K-Ras4B, H-Ras and TC21. These structures suggest that all peptide substrates adopt a common binding mode in the FTase and GGTase-I active site. Unexpectedly, while the X residue of the Ca1a2X motif binds in the same location for all GGTase-I substrates, the X residue of FTase substrates can bind in one of two different sites. Together, these structures outline a series of rules that govern substrate peptide selectivity; these rules were utilized to classify known protein substrates of CaaX prenyltransferases and to generate a list of hypothetical substrates within the human genome.