Enzyme Flexibility and the Catalytic Mechanism of Farnesyltransferase

J. Phys. Chem. B 2008, 112, 8681–8691

8681

Enzyme Flexibility and the Catalytic Mechanism of Farnesyltransferase: Targeting the Relation Se´rgio F. Sousa, Pedro A. Fernandes, and Maria Joaõ Ramos* REQUIMTE, Departamento de Quı´mica, Faculdade de Cieˆncias, UniVersidade do Porto, Rua do Campo Alegre, 687, 4169-007 Porto, Portugal ReceiVed: NoVember 27, 2007; ReVised Manuscript ReceiVed: May 14, 2008

Farnesyltransferase enzyme (FTase) is an interesting target for anticancer therapy that has been the subject of particular attention over the past decade. However, despite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This work describes the application of molecular dynamics simulations to the study of enzyme flexibility in the 4 key intermediate states formed during the FTase catalytic mechanismsFTase resting state, binary complex (FTase-FPP), ternary complex (FTase-FPP-Peptide), and product complex (FTaseProduct)sthereby covering the main states in the mechanistic pathway of this mysterious enzyme, while relating, dissecting, and exploring, in minute detail, the set of structural and dynamical changes taking place with FPP binding, peptide coordination and product formation. This study reveals the existence of a series of variational patterns involving the mechanistic events taking place at the active site of the enzyme, increasing in magnitude away from the active-site, demonstrating that relatively small-scale events such as substrate binding or product formation cause minor changes at the neighboring residues and corresponding helices, but ultimately induce much more dramatic effects on the more external regions of the enzyme. Introduction Farnesyltransferase (FTase) is a zinc enzyme that consists of a 48 kDa R-subunit and a 46 kDa β-subunit1–3 and that has been the subject of particular interest in anticancer research over the past decade.4–6 This enzyme promotes the addition of the isoprenoid farnesyl group from farnesyl diphosphate (FPP) to a cysteine residue of a protein substrate containing a typical CAAX motif at the carboxyl terminus, where C represents the cysteine residue that is farnesylated, A is an aliphatic amino acid, and X represents the terminal amino acid residue, usually alanine, serine, methionine, or glutamine.7 The list of known substrates of FTase comprises the H-, N-, and K-Ras proteins, nuclear lamins A and B, the γ subunit of heterotrimeric G-proteins, and several proteins involved in visual signal transduction.8,9 Initial interest in FTase arose from the discovery that farnesylation is absolutely necessary for oncogenic forms of Ras proteins to transform cells;10–12 Ras proteins have been implicated in around 30% of all human cancers.13,14 Targeting FTase thus became for some years the Holy Grail of anticancer drug design and development,15 with an absolute plethora of patents describing FTase inhibitors (FTIs) published6 and several drugs moving into clinical testing.16–20 Despite, the very promising initial results, the outcome of phase II and phase III clinical trials was rather disappointing, with the most advanced FTIs failing to demonstrate antitumor activity in Ras dependent cancers.21,22 In fact, K-Ras, the major most frequently mutated form of Ras in human cancers, is able to bypass the FTI blockade through cross-prenylation by the related enzyme geranylgeranyltransferase I (GGTase I).23–25 These conclusions, together with the good results obtained by FTIs in several nonras dependent cancers,26–28 indicating that farnesylated proteins other than Ras must also play a role in the biological consequences * Corresponding author. E-mail: [email protected].

of FTI treatment, have launched the grounds for a new and exciting era in FTIs research and development, still in its infancy.15 However, the precise target of FTase inhibition that is responsible for the observed antitumor activity is still obscure. In addition, FTase inhibitors have also been explored in other noncancer related illnesses, including malaria,29–32 Chagas disease,33,34 African sleeping sickness,35,36 Toxoplasmosis37 and Leishmaniasis.33 Despite the enormous curiosity surrounding FTase inhibition and the vast research in the field, several important aspects of the catalytic mechanism of FTase have remained unexplained or are still a matter of dispute.38 A detailed knowledge of the precise catalytic and inhibition mechanisms of FTase would be a key step in the path toward mastering FTase activity. In this study, we examine in detail several dynamic properties associated with the flexibility of the enzyme and related both with substrate coordination and with catalysis itself, by comparing the behavior exhibited by the four key intermediates formed in the FTase catalytic cyclesFTase resting state, binary complex (FTase-FPP), ternary complex (FTase-FPP-Peptide), and product complex (FTase-Product)sthereby covering all of the main stationary states on the mechanistic pathway of this mysterious enzyme, using three sets of parameters, committed to the AMBER force field,39,40 specifically designed to allow a reliable treatment of three different Zn coordination spheres formed during catalysis, and previously validated against 14 FTase crystallographic structures, EXAFS data, DFT, and QM/MM theoretical calculations.41 The analysis performed here allows a relationship to be drawn between specific catalytic alterations taking place at the activesite (such as FPP binding, peptide coordination, or product formation), and the more global structural and dynamic changes involving the several amino acid residues, helices and subunits that constitute the full enzyme, disclosing important links that would not have been identifiable by a simple static analysis of

10.1021/jp711214j CCC: $40.75  2008 American Chemical Society Published on Web 06/24/2008

8682 J. Phys. Chem. B, Vol. 112, No. 29, 2008 the existing X-ray crystallographic structures. The global view drawn here, encompassing both structural and dynamical aspects of FTase activity enables a detailed understanding of FTase as a fully integrated molecular machine tuned for catalysis. Methods Molecular Dynamics Simulations. For each of the 4-key intermediate states of the FTase catalytic cycle models were prepared from the crystallographic structures with the best resolution for each state: 1FT1 (Enzyme resting state);42 1FPP (binary complex FTase-FPP);43 1JCR (ternary complex FTaseFPP-Peptide);44 and 1KZP (product complex).45 The peptide substrate CVIM from the structure 1D8D46 was later modeled into the 1JCR active site, at the position of the nonsubstrate peptide inhibitor CVFM. The study was limited to the analysis of the four catalytic intermediates outlined above, because only for these states are high resolution X-ray structures available. The existence of such structures is vital for the use of detailed and realistic FTase models at the atomic level. Conventional protonation states for all amino acids at pH 7 were considered; that is, all of the glutamic acid and aspartic acid side chain carboxyl groups were considered deprotonated, whereas the arginine and lysine side chain groups were considered protonated. The histidine amino acid residues were all treated as neutral, with the corresponding protonated nitrogen atoms being chosen on the basis of the possible interactions with the local enzymatic environment. Na+ ions were placed using the Leap program to neutralize the highly negative charges (ranging from -20 to -24) of the systems. The systems were then placed in rectangular boxes containing TIP3P water molecules, with a minimum distance of 15.0 Å of water molecules between the enzyme and the box side, resulting in systems comprised of ca. 140 000 atoms. The AMBER 8.047 molecular dynamics package was used in all the molecular dynamics simulations performed. Three different sets of parameters41 specifically designed to allow a reliable treatment of three different Zn coordination spheres formed during catalysis and based on DFT (B3LYP) and molecular mechanical calculations,48–51 crystallographic data,42,44,45 extended X-ray absorption fine structure (EXAFS) results,52 and on several other recent mechanistic studies53–56 were applied to the four key states of the FTase catalytic pathway. The choice to use three different parameter sets to represent the three different metal coordination spheres formed during catalysis was made due to the influence that the nature and identity of the ligands of the metal sphere have on the bondlengths, angles, and charges involving the metal atom. All of the bonds containing Zn and all of the angles where Zn was the central atom were computationally parametrized (with B3LYP/SDD) for each of the three Zn coordination spheres. The angles for which Zn was a terminal atom were considered independent of the environment and, hence, transferable and were obtained from other Zn enzymes in the literature.57,58 All of the dihedral parameters involving the Zn-ligand interactions were set to zero, a standard approximation when a bonded potential is used to describe this type of systems, to avoid any unnatural decrease in the mobility of the residues surrounding the metal atom.59–66 The van der Waals parameters for the Zn atom in the three environments considered were taken from a set of studies with the enzyme alcohol dehydrogenase, in which a bonded potential was also used,58,67,68 as van der Waals parameters are normally transferable between roughly similar environments. Therefore, the van der Waals parameters for the

Sousa et al. atoms directly coordinating to the Zn ion were assigned from the closest existing AMBER atom types in the standard AMBER force fields.39,40 RESP charges69,70 for the three Zn coordination spheres were derived at the B3LYP/6-311++G(3df,2pd) level. Even though the standard AMBER force fields39,40 were parametrized with HF/6-31G*, B3LYP is considered a far better method to calculate RESP charges in systems involving metals63 and is commonly used,63,71–74 as HF/6-31G* generally underestimates much of the charge transfer effect involving the metal atom.63,72 The three sets of parameters were validated for the four main intermediates of the FTase catalytic cycle against X-ray crystallographic data42,44,45 and extended X-ray absorption fine structure (EXAFS) results.52 The full parametrization and validation protocols are described in more detail in the literature.41 All systems were subjected to a 4-stages refinement protocol using the SANDER module of AMBER 8.0, in which the constraints on the enzyme (50 kcal/mol/Å2) were gradually removed. The constrained portion of each system in the three initial stages is comprised of (1) full enzyme, (2) all heavy atoms, and (3) backbone alfa carbons and nitrogens. This process ended in a full energy minimization (4th stage) until the rms gradient was smaller than 0.02 kcal mol-1 Å-1. MD simulations on the four systems were carried out using the SANDER module of AMBER 8.0, and considering periodic boundary conditions to simulate a continuous system. The SHAKE algorithm75 was applied to fix all bond lengths involving a hydrogen atom, permitting a 2-fs time step. The particle-mesh Ewald (PME) method76 was used to include the long-range interactions, and a nonbond-interaction cutoff radius of 10 Å was considered. Following a 40 ps equilibration procedure, a 10 ns MD simulation was carried out for each of the four systems (a total of 40 ns of MD simulation) at 300 K, using Berendsen temperature coupling and constant pressure (1 atm) with isotropic molecule-based scaling. The MD trajectory was sampled every 0.2 ps. All of the MD results were analyzed with the PTRAJ module of AMBER 8.0. The rootmean-square fluctuation (RMSF) in relation to the average structures within each simulation was also calculated for the full enzyme and for each subunit, helix, and amino acid residue, considering all the CR atoms. The average distances between the CR atoms of all of the main pairs of helices of subunits R and β were also calculated. RMSF values and average distances between pairs of helices were calculated for each set of 500 ps in the 10 ns of simulation time. The values obtained for the several 500 ps intervals were used to calculate the error associated to the corresponding average values. The initial 500 ps of simulation was discarded in the calculation of the RMSF values and of the average distances between pairs of helices. Calculation of the Conservation Grades. The ConSurfHSSP database77 was used to obtain estimated conservation grades for each amino acid position along the FTase sequence. This database contains conservation scores calculated by the Rate4Site algorithm78 for each amino acid position within each three-dimensional protein structure present in the related homology-derived secondary structure of proteins (HSSP) database,79,80 which holds multiple sequence alignments for three-dimensional protein structures from the Protein Data Bank. A total of 28 amino acid sequences were considered for the R-subunit, whereas for the β-subunit the total number was of 78 sequences. The ConSurf-HSSP database may be accessed at http://consurfhssp.tau.ac.il or via the PDBsum Web site http://www.ebi.ac.uk/ thornton-srv/databases/pdbsum/.

Enzyme Flexibility and FTase

J. Phys. Chem. B, Vol. 112, No. 29, 2008 8683

TABLE 1: RMSF by Subunit in FTase, Average Root Mean Square Fluctuation for the r- and β-Subunits of FTase RMSF CR (Å) average RMSF CR highly conserved residuesa (%) resting state binary complex ternary complex product complex (Å) subunit total number of residues R β

377 437 a

25 21

1.01 ( 0.04 0.81 ( 0.04

0.92 ( 0.03 0.79 ( 0.02

0.62 ( 0.03 0.54 ( 0.02

0.83 ( 0.04 0.65 ( 0.02

0.85 ( 0.03 0.70 ( 0.03

Residues with a conservation score of 7 or higher, in a 1-9 scale (9 ) conserved and 1 ) variable).

Figure 1. Schematic representation of the topology of subunits R and β in FTase, with major emphasis being given to the several helices that constitute the enzyme and to the percentage of highly conserved residues. These residues are those with a conservation score of 7 or higher, in a 1-9 scale (9 ) conserved and 1 ) variable).77 The numbers indicated represent the initial and final residues of each helix.

Results and Discussion Subunits r and β. FTase is a heterodimeric Zn enzyme that contains two very different subunits. Subunit R is composed of 377 amino acids,2,3 has a molecular mass of approximately 48 kDa, and is identical to the R-subunit of the related enzyme geranylgeranyltransferase I (GGTase-I).81 The β-subunit of FTase is distinct from the one of GGTase I but has an overall amino acid similarity of 30%.82 It has a lower molecular mass than subunit R (46 kDa) but contains more amino acids: a total of 437. The active site Zn ion is located at the interface of the two subunits at the top of the R-R barrel, in a junction between an interfacial hydrophilic surface groove that runs parallel to the rim of the R-R barrel and an almost orthogonal deep cleft in the β-subunit.42 The isoprenoid moiety of FPP binds in an extended conformation44 to this funnel-shaped hydrophobic cavity, with the diphosphate moiety packed in a positively charged cleft at the top of this cavity near the subunit interface and adjacent to the catalytic Zn ion.83 Zn is coordinated to the side chains of three residues from the β subunit: Asp297β (mono or bidentate coordination),48,84 Cys299β, and His 362β. The R-subunit contacts the β-subunit barrel around half-its circumference at the upper, open end of the barrel, placing several of their residues near the active-site Zn and forming part of the FPP binding site.43 From the analysis of the average RMSF for the 4 states studied (Table 1), it can be seen that the R-subunit exhibits a

higher degree of flexibility (higher RMSF) than its β counterpart (average RMSF of 0.85 and 0.70 Å, respectively), which is in agreement with the structural characteristics and global architecture of each subunit. The crescent-shaped seven-helical hairpin domain defined by the R-subunit offers a relatively larger surface of contact with the solvent than the one presented by the R-R barrel structure of the β-subunit. Hence, the R-subunit contains more amino acid residues with a free side chain than the β-subunit, which directly translates into the RMSF patterns observed for the two chains. A comparison of the RMSF values allows one to analyze the behavior of each structural element in the four FTase catalytic states studied. Our results show that the flexibility of both subunits decreases during the catalytic cycle of FTase (Table 1). Both subunits experience a moderate flexibility decrease with FPP binding, which is followed by a very pronounced change upon peptide binding. Product formation partially restores the flexibility of both subunits but significantly below the level observed in the absence of the peptide substrate. Interestingly, the coordination of FPP to the hydrophobic cavity located in the center of the R-R barrel defined by the β-subunit actually induces more significant changes in the general flexibility of the R-subunit,than in the β-subunit itself. The R-subunit also experiences, in general, more significant alterations on the average RMSF values than the β-subunit. These observations highlight the importance of the R-subunit in

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TABLE 2: RMSF by Group of Helices in FTase, Average Root Mean Square Fluctuation for the Internal and External Helices of the r-Subunit and for the Core and Peripheral Helices of the β-Subunit of FTase RMSF CR (Å) chain Α Α β β

type internal external core peripheral

helices

resting state binary complex ternary complex product complex average RMSF CR (Å)

3R, 5R, 7R, 9R, 11R, 13R, 15R 0.78 ( 0.02 2R, 4R, 6R, 8R, 10R, 12R, 14R 0.94 ( 0.03 3β, 5β, 7β, 9β, 11β, 13β 0.63 ( 0.02 2β, 4β, 6β, 8β, 10β, 12β 0.73 ( 0.03

substrate binding/product formation. In addition, they raise interesting questions concerning the behavior of the related enzyme GGTase I, which shares an identical R-subunit81,82 and has a structurally similar β-subunit but catalyzes the transfer of a 20 carbon isoprenoid group (instead of the 15 carbon farnesyl group characteristic of farnesylation) from geranylgeranyl pyrophosphate to peptide substrates with a CAAX motif in which X is typically leucine or phenylalanine.7,85,86 Groups of Helices. FTase is mainly composed of R-helices (a total of 29). Given the particular architecture of the R-subunit (a double layered crescent-shaped seven-helical hairpin domain enveloping part of the β-subunit), the 15 R-helices that compose this subunit can be grouped into two main types (Figure 1): internal helices and external helices. All helices from a given group are roughly parallel to each other. There are 7 internal R-helices (3R, 5R, 7R, 9R, 11R, 13R, and 15R) and 7 external R-helices (2R, 4R, 6R, 8R, 10R, 12R, and 14R). The first are in direct contact with the β-subunit, whereas the second are disposed around the internal helices and support most of the interactions of the R-subunit with the solvent. The β-subunit of FTase comprises 14 R-helices that are disposed in an R-R barrel motif, blocked at one end by helix 14β and solvent accessible at the other end (Figure 1). Most of the remaining helices can be divided into two groups of 6 R-helices: the core helices (3β, 5β, 7β, 9β, 11β, and 13β), which constitute the inner portion of the barrel; and the peripheral helices (2β, 4β, 6β, 8β, 10β, and 12β), which define the outside of the barrel. In addition to a schematic representation of the topology of both subunits, Figure 1 also represents the percentage of highly conserved residues that constitute each helix. Highly conserved residues are considered those that have a conservation score of 7 or higher, in a 1 to 9 scale (where 9 is conserved, and 1 is variable),77 calculated from the conservation grades given by the Rate4Site algorithm.78 From the analysis of Figure 1, it can be seen that the internal helices of the R-subunit and the core helices of the β-subunit are significantly more conserved than the external and peripheral helices, which is in agreement with the fact that the internal and core helices are responsible for most of the interactions with the two substrates and with the product molecule, and account for most critical issues like substrate orientation and selectivity. Another interesting aspect that can be inferred from this figure is that a very high percentage of catalytically important residues are not included in any of the helices. Examples include the Zn ligands Asp297β and Cys299β, the pyrophosphate stabilizing residues Lys164R, His248β, Arg291β, Lys294β, Tyr300β,55 and the Asp352β residue that has been suggested to coordinate a catalytically important Mg ion in the chemical step of the farnesylation mechanism.53 Several of the FTase critical residues are located in loops or in more disordered regions of the enzyme and not in the more traditionally well-defined secondary-structural elements (R-helices and β-sheets). Table 2 presents the RMSF values (of the CR atoms) for these four groups of helices in the four catalytic states of FTase: internal and external helices from the R-subunit and core and

0.70 ( 0.02 0.94 ( 0.04 0.52 ( 0.02 0.67 ( 0.02

0.50 ( 0.02 0.58 ( 0.02 0.42 ( 0.01 0.48 ( 0.02

0.67 ( 0.02 0.78 ( 0.03 0.47 ( 0.01 0.61 ( 0.01

0.66 ( 0.03 0.79 ( 0.05 0.51 ( 0.02 0.62 ( 0.02

peripheral helices from the β-subunit. As expected, the set of internal helices of the R-subunit and the set of core helices of the β-subunit exhibit a much smaller flexibility than their more external counterparts (the group of external helices of the R-subunit and the group of peripheral helices in the β-subunit). All of the four sets of helices react to FPP binding, peptide coordination, and product formation. The set of internal helices of the R-subunit and the set of core helices of the β-subunit exhibit a more significant decrease in the flexibility with FPP binding than the other two sets. However, with peptide coordination the most dramatic flexibility drops take place on the more external sets of helices of both subunits. Product formation increases the flexibility of all four sets of helices, a change that is more pronounced also for the more external sets. These results highlight the existence of global alterations in the flexibility of the enzyme during catalysis, in particular in some regions located significantly away from the active-site. This behavior would not be expected from the static picture given from the available X-ray structures. In fact, all of the available crystallographic structures for the several catalytic intermediates present only but small structural differences between themselves. Hence, the structure is globally maintained, but the flexibility of the several FTase components varies appreciably during catalysis. Global RMSF Patterns. Figure 3 represents the RMSF of the CR atoms of all the residues in the R and β-subunits in the four catalytic states of FTase. The figure also represents the relative location of all R-helices in the two subunits of the enzyme. The results show that the more flexible regions of the enzyme correspond to regions interconnecting the several

Figure 2. Representation of the average flexibility of each residue on the 4 catalytic states of FTase, mapped into the 1JCR crystallographic structure44 of the FTase ternary complex. (A) The full enzyme, with the R-subunit represented in tubes and the β-subunit represented as a surface; (B) active-site detail; and (C) the R-R barrel of the β-subunit.



Figure 3. RMSF of the CR atoms of all of the residues in the R- and β-subunits of FTase, in the resting state, binary complex, ternary complex, and product complex, with indication of all of the helices and connecting regions and the corresponding percentages of highly conserved residues (the ones with a conservation score of 7-9 in a 1-9 scale) of each one.

R-helices. In addition to the regions on both subunits closer to the N- and C-terminus, the most flexible regions of both subunits are the ones between helices 13R and 14R (in the R-subunit), and between helices 1-2β and 13-14β (in the β-subunit). Both regions are mainly comprised by residues with a low conservation score (Figure 3). Several other regions with a relatively high flexibility are also poorly conserved. Examples include the regions between the helices 11-13R, and 3-4β. Important exceptions are the regions between helices 4-5R, and in particular between helices 12-13β, highly flexible regions comprised by more than 45% of highly conserved residues. The region between helices 12-13β includes several highly conserved residues, like Gln344β, Gly348β, Gly349β, Asp352β, Lys353β, Pro354β, and Asp359β, some of which have been extensively studied by mutagenesis,53,87,88 and comprises an important portion of the active-site cavity. Globally, the results presented in Figure 3 also show that the more flexible helices in FTase (helices 4R, 12R, 14R, 1β, 2β, 4β, and 6β) are typically comprised of residues with a lower conservation score, are external helices in the case of the R-subunit or peripheral helices in the case of the β-subunit (Figure 1), and are mainly comprised of residues located in the first or final third of the amino acid sequence of each subunit. The less flexible helices (4R, 7R, 9R, 11R, 3β, 5β, 9β, and 13β), on the other hand, are more conserved and are located in the internal half of the R-subunit or are part of the core helices in the β-subunit. Figure 2 represents the average flexibility of each residue on the 4 catalytic states of FTase, mapped into the 1JCR crystallographic structure44 of the FTase ternary complex and partially illustrates the conclusions obtained in this section. These representations further show that the active-site region, including the Zn coordination sphere and the amino acid residues that account for most of the interactions with the two substrates, has a very low average flexibility. Figures 4 and 5 represent the change in RMSF that takes place with FPP binding, peptide coordination and product formation for the R- and β-subunits, respectively, with values calculated from the difference between the RMSF of the initial and final states for each of these changes. For example, the RMSF for FPP binding was obtained from the difference between the RMSF of all of the residues in the FTase binary complex and in the FTase resting state. These representations

illustrate the regions of the enzyme where the changes in flexibility are more significantly altered by the coordination of the two substrates or the formation of the product. From Figures 4 and 5, it can be observed that these changes in flexibility are also more pronounced in the regions interconnecting the several R-helices. Some of these regions contain a percentage superior to 75% of highly conserved residues and are going to be the subject of particular attention. These are the regions between the helices 4-5R (75% of highly conserved residues), 10-11β (89%), and 12-13β (71%). 4-5r. The moderately flexible region connecting helices 4 and 5R contains 75% of highly conserved residues and experiences a marked decrease in flexibility with FPP binding. Peptide coordination causes an additional decrease in the flexibility of this region (that is maximal for the 4R helix), which is followed by an important increase in flexibility which takes place upon product formation. This loop connects helices 4R and 5R through the internal side of the enzyme (Figure 1), defining part of the active site and interacting partially with the highly conserved helices 3R and 7R, and contains three highly conserved residues: Gln162R, Lys164R, and Asn165R. Lys164R is one of the most important residues of FTase and has been the subject of several mutagenesis studies,54,89,90 having been directly implicated in the stabilization of the pyrophosphate moiety of the FPP substrate. Our results are in agreement with the proposed role for Lys164R and the relative position of this residue in the enzyme. The binding of the FPP substrate and the formation of a strong interaction between Lys164R and one of the oxygen atoms from the pyrophosphate part of FPP constrains the flexibility of this region. Peptide entrance, with the conformational change of the FPP substrate, does not alter significantly the flexibility value. Finally, with the transfer of the farneysl group to the cysteine from the peptide substrate, upon product formation, the pyrophosphate group leaves the enzyme, and the flexibility of this region rises again. Hence, our results suggest that this region reacts directly to the changes taking place at the active-site. 10-11β. A very important part of the enzyme is the region between helices 10-11β, which encompasses much of the active site residues, and is comprised by almost 90% of highly conserved residues, including the Zn binding Asp297β and Cys299β, and several other residues that have been implicated


Sousa et al.

Figure 4. Change in the RMSF for each amino acid residue in the R-subunit that takes place with FPP binding, peptide coordination, and product formation, with indication of all of the helices and connecting regions and the corresponding percentage of highly conserved residues (the ones with a conservation score of 7-9 in a 1-9 scale) of each one.

in the stabilization of the oxygen atoms from the pyrophosphate group from FPP, as Arg291β, Lys294β, and Tyr300β.54 In this region, FPP binding causes an increase in flexibility, which is followed by a marked decrease upon peptide coordination and a very slight increase with the formation of the Zn-bound product. This region is located in a key-position in the core of the enzyme, establishing direct contact with the pyrophosphate moiety of the FPP in the ternary complex but also with an important portion of the farnesyl part of FPP. It also interacts partially with the peptide substrate, particularly at the Zn-bound Cys residue. In addition, this region interacts directly with several highly conserved key-helices like the 7R, 9R, 11R, 9β, 12β 13β, and 14β and with other nonconserved helices such as the 13R and the 1β. The residues in the region connecting helices 10 and 11β where the changes in flexibility are more pronounced are the ones directly interacting with the pyrophosphate moiety, the Zn-bound peptide thiolate, and helix 1β. The marked decrease in flexibility observed upon peptide coordination is in agreement with the conformational change suffered by the FPP molecule with peptide entrance, which positions FPP in close contact with several residues of this region. Several regions on the enzyme that interact with this region also follow similar trends. Altogether, our results indicate that this region reacts to FPP binding, peptide coordination, and product formation and induces changes in the flexibility of several more distant regions of the enzyme. 12-13β. The region in the enzyme between helices 12 and 13β is 71% composed of highly conserved residues and comprises also an important part of the enzyme’s active-site.

Among the most conserved residues in this region are the Gln344β, Gly348β, Gly349β, Asp352β, Lys353β, Pro354β, and Asp359β. FPP binding induces an increase on the flexibility of approximately half of the residues that constitute this region, whereas the flexibility of the other half-is decreased. However, with peptide coordination, the flexibility of the entire region is diminished. Product formation leads to a very important increase in the flexibility of the first half of the region (residues 342 to 352β). The most dramatic changes in flexibility in the 12-13β region take place at the residues directly interacting with the Zn coordination sphere and with helix 1β. Mutagenesis studies at the Asp352β position have shown to drastically alter the Mg2+ dependence of the catalytic step of FTase, without significantly altering the coordination affinity of both substrates or the velocity of the reaction in the absence of Mg2+.53 These results have been interpreted as indicative of Asp352β coordination to a catalytically important Mg2+ ion. This residue is one of the most internal residues of this region, and establishes very close contact with the 10-11β region, and in particular with Asp297β, a very important Zn ligand, known to circle between monodentate and bidentate coordination during the FTase catalytic pathway.48 The very marked decrease in flexibility observed with peptide coordination is coherent with the change from a state where Asp297β is able to alternate between mono to bidentate coordination, to a state where only the bidentate form is possible.48,49 The Asp352β residue thus reacts to the alterations taking place at the Zn center. Mutagenesis studies on Asp359β have suggested a possible role in Zn coordination, even though this residue is not directly



Figure 5. Change in the RMSF for each amino acid residue in the β-subunit that takes place with FPP binding, peptide coordination and product formation, with indication of all the helices and connecting regions and the corresponding percentage of highly conserved residues (the ones with a conservation score of 7-9 in a 1 to 9 scale) of each one.

bound to the metal ion.87 In fact, in the available X-ray crystallographic structures, Zn is at a distance of around 7 Å from the Asp359β residue. Our calculations show that Asp359β is one of the least flexible residues in the enzyme. They further demonstrate that this residue experiences one of the smallest changes in terms of RMSF in the 12-13β region with ligand binding and product formation. This residue occupies a keyposition at the loop, which marks the beginning of helix 13β that passes directly under the Zn coordination sphere, contains the Zn ligand His362β and interacts with Zn ligands Asp297β and Cys299β. It is thus possible that a mutation at the 359β position would have a direct effect on helix 13β, indirectly affecting also the Zn coordination sphere. So, on the whole, our results also show that the 12-13β region reacts to FPP binding, peptide coordination, and product formation and further influences alterations in the flexibility of other regions on the enzyme. Structural Changes. To further illustrate the set of alterations induced in the enzyme by FPP binding, peptide coordination, and product formation, we have analyzed also the structural alterations taking place during the FTase catalytic cycle. Taking into consideration the particular architecture of the FTase enzyme, attention was dedicated to the analysis of the average distance (calculated from the MD simulations) between the CR atoms from different pairs of R-helices. The R-R barrel in the β-subunit comprises the most important part of the active-site of FTase. Figure 6 illustrates, in a simplified form, the overall topography of the R-R barrel

in the β-subunit and the structural changes taking place with FPP binding, peptide coordination and product formation. In this figure, special emphasis was dedicated to the representation of the average distances between the CR atoms in each pair of parallel helices (core-peripheral) and of the average distances between the CR of opposite helices located at the core of the R-R barrel. The results show that FPP binding enlarges the distances between the several core helices of the R-R barrel in the β-subunit in a process that presses these helices against their peripheral pair helices. Hence, the distance between helices 3 and 2β, 7 and 6β, 9 and 8β, and 11 and 10β falls, whereas the distance between opposite helices at the core of the barrel increases. Coherent with these structural alterations taking place, the flexibility of helices 3β, 5β, 7β, 9β, 11β, and 13β also falls (see Figure 5). The peptide substrate binds in an extended conformation and interacts strongly with helices 13β (the Zn binding portion of the peptide, i.e., the Cys residue) and 5β (the carboxyl-terminus of the peptide, i.e., the X group of the CAAX motif). Hence, the distances between helices 13β and 12β, and 5β and 4β also decrease. Globally, for helices 7β, 11β, and particularly 3β, the interactions of the substrate molecules with the core helices of FTase are less pronounced in the ternary complex than in the binary complex. Hence, the distance between these helices and their peripheral pairs increases (Figure 6). Product formation causes less dramatic changes in the overall topography of the R-R barrel than FPP binding or peptide


Sousa et al.

Figure 6. Simplified representation of the overall topography of the R-R barrel of the β-subunit and the structural changes taking place with FPP binding, peptide coordination and product formation. The values refer to the average distances in Angstroms between the CR atoms of each pair of helices, and to the corresponding changes with FPP binding, peptide coordination, and product formation, calculated from the MD simulations performed.

coordination. Only marginal changes in the distances between different pairs of helices were observed to take place for the R-R barrel of the β-subunit with product formation, in agreement with the small variation observed in flexibility of the residues in this subunit. In the R subunit, the distances between successive pairs of external helices (2R, 4R, 6R, 8R, 10R, 12R, and 14R) tend to react in the opposite direction to the changes taking place between successive pairs of internal helices. Hence, while the distance between helices 5 and 7R increases, the distance between helices 4 and 6R decreases (see Figure 7). The very same trend can be observed for all of the central helices of the R-subunit of FTase (from helix 5R to 13R) with FPP binding and in most cases with peptide coordination and product formation (Figure 7). Interestingly, a small change in the distance between two successive internal helices corresponds to a much more considerable variation in the distance between the corresponding external helices (in some cases 4- or 5-fold). This shows that a small change at the active site, such as the one caused by the coordination of a small substrate molecule, can cause much more significant changes in a set of surrounding structural elements that ultimately induce more dramatic changes in the more external structural elements enveloping the enzyme. Hence, a small change at the active-site is amplified to the full enzyme. This conclusion is in agreement with Figure 8, which represents

the maximum change in flexibility (in terms of the RMSF) for each residue on the 4 catalytic states of FTase, showing that the regions in the enzyme where the changes in the RMSF are the highest (considering the set of changes taking place with FPP binding, peptide coordination and product formation) are located near the enzyme surface, whereas the region surrounding the active-site experiences only minor alterations. Conclusions The set of results obtained in this study has enabled a fully integrated analysis of the structural and dynamical behavior of FTase in the four key-states formed during catalysis, bridging the gap between the atomic-level events, such as the ones involving the breaking/formation of bonds at the active site, and the full molecular response, complementing the static snapshot view given from the available X-ray crystallographic structures with a more global picture that takes motion into account. The results show that the main structural and dynamical properties of the active-site of FTase are not significantly altered with FPP binding, peptide coordination, or product formation. However, they do show that these events lead to major changes at the more external regions of the enzyme, particularly in terms of flexibility. Furthermore, they disclose



Figure 7. Simplified representation of the overall topography of the R-subunit and the structural changes taking place with FPP binding, peptide coordination, and product formation. The values refer to the average distances in angstroms between the CR atoms of each pair of helices and to the corresponding changes with FPP binding, peptide coordination, and product formation, calculated from the MD simulations performed.

the existence of a series of variational patterns involving these changes, which increase in magnitude away from the activesite. These effects were observed both in the R-R barrel structure of the β-subunit and in the seven-helical hairpin domain that constitutes the R-subunit. Substrate binding (FPP and the CAAX peptide) and product formation induce small changes in the neighboring residues and corresponding helices, which then cause higher magnitude effects on other regions of the enzyme. Hence, a relatively small change at the active-site is amplified into a much more significant alteration at the external surface of the enzyme. These conclusions can be of great interest in FTase research, since this enzyme is thought to act on a great variety of proteins, holding a multitude of different characteristics, but all having in common a typical CAAX motif. The type of structural and dynamic alterations identified in this study are biologically significant, and may affect protein-protein interactions between FTase and its protein substrates or with other protein

partners like the set of enzymes involved in the subsequent steps of post-translational modification of the CAAX substrates, in which the AAX portion is proteolytically removed and the newly formed farnesylated cysteine residue is methyl esterified.91 The results further highlight the importance of a set of highly conserved key-regions on the enzyme. Most of these regions are located in structurally disordered regions connecting different helices. Examples include 1-2R, 4-5R, 7-8R, 2-3β, 4-5β, 10-11β, and the 12-13β regions. The magnitude of the changes induced by the alterations taking place at the active-site is particularly large for a set of poorly conserved helices located considerably away from the active site, in particular for helices 1R, 4R, 10R, 12R, 1β, and 4β. The interaction of potential FTase inhibitors or natural substrates with these regions on the enzyme should be taken into account for a better understanding of FTase activity and for the development of more specific inhibitors.


Figure 8. Representation of the maximum change in flexibility (in terms of the RMSF) for each residue on the 4 catalytic states of FTase, mapped into the 1JCR crystallographic structure44 of the FTase ternary complex. (A) The full enzyme, with the R-subunit represented in tubes and the β-subunit represented as a surface; (B) active-site detail; (C) surface representation of the full enzyme.

The results further reveal that peptide coordination causes a more significant impact on the structural and dynamical properties of FTase than FPP binding, and that the changes taking place with product formation are much less considerable. However, even a small change at the active-site, like the one taking place with product formation has a significant impact on the structure and flexibility of the more external regions of the enzyme. Acknowledgment. We thank the FCT (Fundaçaõ para a Cieˆncia e a Tecnologia) for financial support (POCI/QUI/61563/ 2004). Se´rgio Filipe Sousa also acknowledges the FCT for a doctoral scholarship (SFRH/BD/12848/2003). Supporting Information Available: Graphic representations of the root mean square deviation (rmsd) variation in the molecular dynamics simulations performed for the four systems considered: FTase resting state, binary complex, ternary complex, and product complex. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Reiss, Y.; Goldstein, J. L.; Seabra, M. C.; Casey, P. J.; Brown, M. S. Cell 1990, 62, 81. (2) Chen, W. J.; Andres, D. A.; Goldstein, J. L.; Brown, M. S. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 11368. (3) Chen, W. J.; Andres, D. A.; Goldstein, J. L.; Russell, D. W.; Brown, M. S. Cell 1991, 66, 327. (4) Johnston, S. R. Lancet. Oncol. 2001, 2, 18. (5) Sebti, S. M. Oncologist 2003, 8, 30. (6) Huang, C. Y.; Rokosz, L. Expert. Opin. Therap. Patents. 2004, 14, 175. (7) Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands, E.; O’Hara, M. B.; Garsky, V. M.; Marshall, M. S.; Pompliano, D. L.; Gibbs, J. B. J. Biol. Chem. 1991, 266, 14603. (8) Schafer, W. R.; Rine, J. Annu. ReV. Genet. 1992, 26, 209. (9) Glomset, J. A.; Farnsworth, C. C. Annu. ReV. Cell. Biol. 1994, 10, 181. (10) Hancock, J. F.; Magee, A. I.; Childs, J. E.; Marshall, C. J. Cell 1989, 57, 1167. (11) Jackson, J. H.; Cochrane, C. G.; Bourne, J. R.; Solski, P. A.; Buss, J. E.; Der, C. J. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3042. (12) Kato, K.; Cox, A. D.; Hisaka, M. M.; Graham, S. M.; Buss, J. E.; Der, C. J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 6403. (13) Dolence, J. M.; Poulter, C. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 5008. (14) Takai, Y.; Sasaki, T.; Matozaki, T. Physiol. ReV. 2001, 81, 153. (15) Rowinsky, E. K. J. Clin. Oncol. 2006, 24, 2981.

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JP711214J

Enzyme Flexibility and the Catalytic Mechanism of Farnesyltransferase

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