AcademicPressisanimprintofElsevier 32JamestownRoad,LondonNW17BY,UK Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands LinacreHouse,JordanHill,OxfordOX28DP,UK 225WymanStreet,Waltham,MA02451,USA 525BStreet,Suite1900,SanDiego,CA92101-4495,USA Firstedition2011 Copyright#2011ElsevierInc. Allrightsreserved Nopartofthispublicationmaybereproduced,storedinaretrievalsystem ortransmittedinanyformorbyanymeanselectronic,mechanical,photocopying, recordingorotherwisewithoutthepriorwrittenpermissionofthepublisher PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRights DepartmentinOxford,UK:phone(+44)(0)1865843830;fax(+44)(0)1865853333; email:permissions@elsevier.com.Alternativelyyoucansubmityourrequestonline byvisitingtheElsevierwebsiteathttp://elsevier.com/locate/permissions,andselecting ObtainingpermissiontouseElseviermaterial Notice Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamagetopersons orpropertyasamatterofproductsliability,negligenceorotherwise,orfromanyuse oroperationofanymethods,products,instructionsorideascontainedinthematerial herein.Becauseofrapidadvancesinthemedicalsciences,inparticular,independent verificationofdiagnosesanddrugdosagesshouldbemade ISBN:978-0-12-415922-8 ISSN:1874-6047 ForinformationonallAcademicPresspublications visitourwebsiteatelsevierdirect.com PrintedandboundinUSA 11 12 13 14 10 9 8 7 6 5 4 3 2 1 Preface Since the discovery of isoprenylation as an enzymatic posttranslational modification in the 1980s, many proteins have been shown to be modified by either a farnesyl or a geranylgeranyl group. In fact, proteomic and database analyses suggest that up to 600 eukaryotic proteins may be isoprenylated. The protein prenyltransferases, FTase and GGTase-I, that catalyze these modifications have been identified and characterized. Isoprenylation occurs at a carboxyl-terminal CaaX sequence, where C is cysteine,‘‘a’’isgenerallyanaliphaticresidue,andXisoneofanumberof amino acids. The Rab family proteins are isoprenylated by a unique Rab prenyltransferase, GGTase-II, at carboxyl-terminal CXC and CC motifs. IsoprenylationofCaaXproteinsisfollowedbyproteolyticcleavageofthe carboxyl-terminal -aaX residues and carboxyl methylation of the newly exposed isoprenylated cysteine residue. The enzymes Rce1 and Icmt are responsible for these postprenylation processes. Interestingly, Rce1 and Icmt are both integral membrane proteins, presenting researchers with an additional set of challenges as they characterize the enzymes and develop inhibitors. We designed a two-volume book series to highlight recent advances in thestudyoftheposttranslationalmodificationsofCaaXandRabproteins. This volume (Vol. 30) is the second (Part B) of the series and covers enzymology and the biological consequences of protein isoprenylation. In addition, several chapters cover the development of inhibitors for the prenyltransferases,Rce1andIcmt,astheseenzymeshaverecentlyemerged as intriguing targets for chemointervention. We believe that these two volumes capture the essence of the extensive information available in the fieldandhighlightsignificantadvances. However,thereareanumberoftopicsthatwecouldnotcoverinthetwo volumes(Vols.29and30)duetospaceconstraints.Forexample,wecould not include a discussion of protein prenylation in pathogenic fungi and trypanosomes and the development of inhibitors for therapeutic applica- tions.Inaddition,wedidnotsufficientlydiscussproteinsthatrecognizeand interact with prenylated proteins and the biological functions of these protein–protein interactions. Future areas to cover should also include expanded discussion on the roles that prenylated proteins play in plants xi xii PREFACE and thepromising useoffarnesyltransferase inhibitorsinthe treatmentof progeria.Theseaswellasothertopicswillbethefocusofafuturevolume onproteinprenylation. We would like to thank the authors for their efforts in producing inter- esting chapters. We would also like to thank Mary Ann Zimmerman and Malathi Samayan of Elsevier for their guidance and encouragement. Finally, we thank Gloria Lee of UCLA for her assistance in communica- tionswithauthorsandthepreparationandeditingofthechapters. ChristineA.Hrycyna MartinO.Bergo FuyuhikoTamanoi September2011 1 The Enzymology of CAAX Protein Prenylation KENDRAE.HIGHTOWERa,b,1 (cid:1) PATRICKJ.CASEYa,b aDepartmentsofPharmacologyandCancerBiology DukeUniversityMedicalCenter Durham,NorthCarolina,USA bDepartmentofBiochemistry DukeUniversityMedicalCenter Durham,NorthCarolina,USA I. Abstract Manyproteinsinvolvedinsignaltransductionandproteintraffickingare posttranslationally modified by the covalent attachment of lipid groups. One form of lipid modification involves attachment of either a 15-carbon farnesylora20-carbongeranylgeranylisoprenoidlipidtoacysteineresidue fourthfromtheC-terminusofthesubstrateprotein.Theattachmentofthe isoprenoidisthe first stepina processing pathway that can include subse- quent proteolysis of three carboxyl-terminal residues, methylation of the freecarboxylgroupoftheresultingC-terminalprenylcysteine,andmodifi- cation with additional lipid molecules. These modifications are necessary for targeting and attachment of these so-called CAAX proteins to the correct membrane as well as for the cellular function of the protein. The focusofthischapterisonthetwoproteinprenyltransferasesresponsiblefor additionoftheisoprenoidtotheCAAXproteinsubstrates. 1 Present address: Molecular Discovery Research, GlaxoSmithKline, Research Triangle Park, Durham,NorthCarolina,USA THEENZYMES,Vol.XXX 1 ISSNNO:1874-6047 #2011ElsevierInc.Allrightsreserved. DOI:10.1016/B978-0-12-415922-8.00001-X 2 KENDRAE.HIGHTOWERANDPATRICKJ.CASEY II. Introduction TheprenylationofCAAXproteinsbecamethesubjectofintensestudy due, in large part, to the cellular functions of these proteins [1,2]. TheenzymesthatcatalyzetheCAAXproteinprenylationreaction,protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I), were identified in the early 1990s [3–5]. Many of the protein substratesfortheseenzymesbelongtotheRassuperfamilyofGTP-binding regulatory proteins (G proteins) and are involved in regulation of cell growth, differentiation, and vesicle transport. In particular, the high inci- dence of activated Ras in human cancers, coupled with the discovery that Ras proteins require farnesylation for biological function, has sparked interest in developing inhibitors of FTase and GGTase-I as chemothera- peutic agents [2,6,7]. The desire to develop enzyme-specific inhibitors, based on either substrate specificity or chemistry, fueled the early studies of the similarities and differences of the closely related FTase and GGTase-I. Several FTase inhibitors have been evaluated in clinical trials as single agents or in combination with other drugs [8,9], and the first GGTase-I inhibitor has also entered clinical evaluation [10]. This review focusesonthebiochemicalreactionsthatproduceCAAX-prenylatedpro- teins.Inparticular,thisworkcoversresearchthathasprovidedinsightinto thesubstratespecificityoftheproteinprenyltransferasesandthechemistry ofthereactionsthattheycatalyze. III. ProteinFTase FTaseisaheterodimerconsistingofanaandabsubunitthatmigrateon SDS-polyacrylamide gels with apparent molecular masses of 48 and 46kDa, respectively, for the mammalian enzymes [3,11]. The discrepancy in the calculated (44kDa) and observed (48kDa) molecular mass of the mammalianasubunitisduetoaproline-richdomainattheamino-terminus oftheproteinthatisnotpresentinasubunitsfromlowerorganisms[12,13]. ThefirstcrystalstructureofFTasewasreportedin1997[14].Theasubunit consists of seven pairs of helices that are configured in antiparallel coiled coils or helical hairpins. The a subunit coiled coils form a crescent shape that wraps around one side of the b subunit, resulting in a substantial subunit interface. The overall structure of the b subunit is an a–a barrel consisting of 12 a-helices. These helices are arranged in two layers of six helicessothattheouterhelicesareantiparalleltotheinnerhelices.Inthe centerofthesehelicesisalargecavity,approximately15A˚ wideand14A˚ 1. CAAXPRENYLTRANSFERASES 3 deep,thatisblockedononeendandopentothesolventontheotherend. Azincion,whichisrequiredforactivitybytheCAAX prenyltransferases [15,16],ispositionednearthetopofthecavityandprovidedevidencethat this large cavity is the active site. Since the publication of the first FTase structure, other structures of substrate and product complexes have con- firmedthelocationoftheactivesiteandprovidedawealthofinformation on the interaction of the substrates and products with both FTase and GGTase-I[17–21]. FTasecatalyzestheformationofathioetherlinkagebetweenthecyste- ineofthecarboxyl-terminalCAAXmotifofaproteinandtheC1position ofthe15-carbonfarnesylgroup.Thefreeenzymehasasingle-bindingsite (cid:3) fortheisoprenoidsubstrateandastable,noncovalentE isoprenoidbinary complexcanbeformed[22,23].WhileFTasecanbindbothfarnesyldiphos- phate(FPP)andthe20-carbongeranylgeranyldiphosphate(GGPP)tightly (for mammalian FTase, K FPP¼(cid:4)5nM and K GGPP¼30(cid:5)100nM) D D [24,25],FPP isthepreferredsubstrateforformationofaprenylatedprod- uct. Crystal structures of FTase with bound FPP and FPP analogues have shown that a hydrophobic cleft formed by 10 aromatic residues is the binding site for the farnesyl group, which is oriented in an extended con- formationwiththediphosphatemoietyatthetopoftheactivesiteandthe carbon tail at the bottom of the active-site cavity [17,18,21]. The farnesyl group interacts with many hydrophobic residues in the active-site cavity, while near the top of the cavity is a region of positively charged residues that forms the binding pocket of the diphosphate moiety of FPP. Several residues form hydrogen bonds with the diphosphate moiety, including Lys164a, His248b, Arg291b, Lys294b, and Tyr300b, and Arg291b, Lys294b, in particular, appear to be important in stabilizing formation of thediphosphateleavinggroup[26]. A general characteristic of proteins modified by FTase is that they contain a carboxyl-terminal Ca a X motif, where the C is a conserved 1 2 cysteine that is the site of prenylation, a and a are usually aliphatic 1 2 amino acids, and the X residue imparts specificity for FTase (for which X is generally Met, Ser, Gln, or Ala) or the related enzyme GGTase-I (for whichXisgenerallyLeu)[2,27].Thepositionofthecysteineasthefourth residue from the carboxyl terminus and the availability of a free thiol are very important for recognition of peptides and proteins as substrates [28,29].TheCa a Xtetrapeptidesequencebindsinanextendedconforma- 1 2 tionintheactivesitewiththecysteineatthetopofthecavitysuchthatthe sulfurcaninteractwiththezincion,thea residuefacingthesolvent,thea 1 2 residue interacting with the enzyme, and the X residue positioned in a hydrophobic pocket [30].High-affinityCAAXsubstratebindingisdepen- dent upon the zinc–sulfur interaction and on interactions between the 4 KENDRAE.HIGHTOWERANDPATRICKJ.CASEY CAAX substrate and the enzyme and bound isoprenoid substrate [25,29,31–33].Screeningofpeptidelibrarieshaverevealedalargenumber of CAAX peptide sequences that can be processed by FTase, including some with Leu at the X residue, hinting that there may be many more farnesylatedproteinsincellsthanpreviouslyestimated[34,35]. ThecatalyticmechanismofmammalianFTaseisfunctionallyorderedso (cid:3) that product formation results from establishment of an initial E FPP binary complex (Figure 1.1). For the mammalian enzyme, the chemical step is not rate limiting in the overall reaction under steady-state condi- tions, since the k value for FTase (0.05s(cid:5)1) is approximately 200-fold cat slowerthantherateconstantforthechemicalstep[25,31].Hence,product releaseistherate-limitingstepintheoverallreaction.Intriguingly,release of the product is almost completely dependent on the binding of an addi- tionalsubstratemolecule,withFPPbeingmostefficientinthisregard[36]; thebiologicalimplicationsofthisfindingarediscussedbelow.Inthecrystal (cid:3) (cid:3) structureofanE product FPPcomplex,thefarnesylchainoftheproductis (cid:3) displaced significantly from its position in the E product complex to a position near the rim of the active site, while the ‘‘second’’ FPP molecule (cid:3) (cid:3) occupiestheFPPsubstrate-bindingsite[18].TheE product FPPstructure PPi E+FPPKD=5nM EFPP+CAAX105M−1s−1EFPP EFarnesyl X CAAX 17s−1 CAAX CAAX 0.05s−1 ECAAX X Farnesyl FPP CAAX FIG.1.1. Kineticschemeforthe(cid:3)reactioncatalyzedbyFTase.Thereactionisfunctionally ordered since formation of the E FPP binary complex leads to product formation. The prenylated CAAX sequence remains bound in the active site following formation of the thioether product, making product release the rate-limiting step in steady-state catalysis. BindingofanadditionalFPPsubstratemoleculepromotesreleaseoftheprenylatedproduct. AdaptedfromRef.[18].(Seecolorplatesectioninthebackofthebook.) 1. CAAXPRENYLTRANSFERASES 5 showsthatseveralresidues,includingTyr93b,Leu96b,andTyr361b,inter- act with the isoprenoid of the product and could affect product release. Indeed, mutation of Tyr361b to Leu in FTase results in an enzyme that is compromised with respect to product release but retains normal substrate bindingandchemistry[37]. ThezincioninFTaseisdirectlyinvolvedinbothsubstrate bindingand catalysis.Thezincionispositionednearthetopoftheactive-sitecavityand is coordinated by three residues from the b subunit, Asp297, Cys299, and His362,andawatermolecule,inadistortedpentacoordinategeometry[14]. The zinc-bound water molecule is displaced by the sulfur of the CAAX motifcysteineuponformationoftheternarycomplex[18,38,39].Interaction ofthe cysteine sulfur withthe zinc lowers the pK of thethiol by approxi- a matelytwopHunits[29,39],suggestingthatthezinc-coordinatedthiolateis presentatphysiologicalpH.Metal-substitutionandpH-dependencestudies offerevidencethatthezinc-thiolateisdirectlyinvolvedinthechemicalstep ofproductformation[31,40].Thezincionmayalsobeimportantfororient- ingthecysteinethiolateforattackontheC1carbonoftheprenylgroup,as (cid:3) (cid:3) suggestedfromacrystalstructureofanE isoprenoid CAAXternarycom- plexwiththezincremoved.Inthisstructure,thecysteineanda residuesof 1 thepeptidewerereorientedsuchthatthesulfurofthecysteinewasdisplaced 9A˚ fromitspositionwhencoordinatedtothemetal[30]. The role of magnesium in the reaction catalyzed by FTase is less well understood than the role of the zinc. Magnesium is required for maximal rates of product formation but is not strictly required for formation of a (cid:3) farnesylated product by FTase, and neither the formation of the E FPP (cid:3) (cid:3) binary complex nor the formation of the E FPP peptide ternary complex appearstobedependentuponmagnesium[11,16,31].Thereisevidencethat magnesium may play a direct role in the transition state of the reaction catalyzed by FTase by coordinating the nonbridging oxygens of diphos- phate to make it a better leaving group, hence facilitating formation of a developing carbocation at C1 of the farnesyl group [18,40]. Alternatively, the magnesium could assist in formation of a catalytically competent ternarycomplex. Much attention has been focused on defining the transition state of FTase and the structural determinants of the chemical step. For FTase, there is evidence for both an electrophilic contribution to the transition state,obtainedfromstudieswithfluoromethylFPPanalogues,andanucle- ophilic contribution, obtained from the metal-substitution and pH studies [31,40,41].Theseresultsaresupportedbytheinabilitytotrapacarbocation intermediate,inversionofconfigurationatC1ofthefarnesylgroupduring thereaction,andana-secondarykineticisotopeeffectnearunity[31,42,43]. Takentogether,theavailabledatasuggestthatthetransitionstateofFTase 6 KENDRAE.HIGHTOWERANDPATRICKJ.CASEY isassociativewithbothelectrophilicandnucleophiliccharacteristics.Inthis transitionstate,thezinc-coordinatedsulfuroftheCAAXcysteinecontrib- utes a partial negative charge and the C1 of FPP contributes a partial positivecharge.FormationofthepositivechargeatC1couldbefacilitated byafore-mentionedstabilizationofthediphosphateleavinggroupthrough coordinationofthenonbridgingoxygensbymagnesiumorinteractionswith residuesintheactivesitesuchasLys164aandTyr300b[44]. IV. ProteinGGTase-I ThesecondoftheCAAXprenyltransferase,GGTase-I,isaheterodimer consistingofa48-kDaasubunitanda43-kDabsubunit[11,45].FTaseand GGTase-I share a common a subunit [11,45,46]. The b subunits of FTase andGGTase-Iaredistinctbuthighlyhomologous,withapproximately30% sequenceidentity[13,47,48]. Much of the interest in the enzymology of GGTase-I has been due to effortstodevelopinhibitorsthatwillblockfarnesylationbyFTasebutnot geranylgeranylationbyGGTase-I,althoughoverthepastdecade,GGTase-I hasemergedasadrugtargetinitsownright[2,49].Whilemanyaspectsof the CAAX prenyltransferases are similar, significant differences do exist that could be exploited to make enzyme-specific inhibitors. Foremost among these differences are those important for substrate binding. As its nameimplies,GGTase-Icatalyzesthetransferofthe20-carbongeranylger- anyl group from GGPP to protein substrates. While GGTase-I modifies manyproteinsintheRassuperfamily,theseproteinssuchasRho,Rac,and Rap,almost exclusivelycontainLeu astheXresidueintheCAAXmotif. Recognitionofthe-aaXsequenceismorerestrictedforGGTase-Ithanfor FTase [50,51], and the a residue and the a –X combination appear to 2 2 impartcontext-dependentrecognitionofCAAXsequencesontheenzymes [20,52]. However, mammalian K-Ras and N-Ras, which have a carboxyl- terminalMet,canbeprenylatedbybothFTaseandGGTase-Iinvitroand, under conditions where FTase is inhibited, in vivo [53–55]. Several other proteins, including the small G protein RhoB (CAAX motif CKVL) and theRas-relatedproteinTC21(CAAXmotifCVIF),havealsobeenshown to be substrates for both FTase and GGTase-I in vitro (RhoB and TC21) and/or in vivo (RhoB) [56–58]. The presence of a polybasic region just upstream of the CAAX motif influences the capacity of a CAAX protein tobemodifiedbyeitherenzyme[59],asdoesthehydrophobicityoftheX residue [51]. These findings suggest that, even if this is not a common occurrenceundernormalconditions,cross-prenylationcouldbeanimpor- tantconsiderationforthetreatmentoftumorswithFTaseinhibitors[60]. 1. CAAXPRENYLTRANSFERASES 7 ThecatalyticmechanismofGGTase-Icloselyresemblesthemechanism of FTase [24,32,61]. It seems almost certain that the transition state of GGTase-I will be similar to that of FTase and that the zinc-bound sulfur actsasanucleophileinthereaction.However,GGTase-Isteady-stateactiv- ityisnotdependentuponthepresenceofmagnesiumions[62].Hence,the role of magnesium, or potentially the ability of specific residues in this regard,inthecatalyticmechanismofGGTase-Iremainstobedetermined. V. Conclusions The past two decades have provided a wealth of information on the biochemistryoftheproteinprenyltransferases.Inparticular,abetterunder- standing of the substrate specificity and catalytic mechanisms of these enzymes has come from detailed biochemical and structural studies. This workhasledtomodelsforthetransitionstateoftheCAAXprenyltransfer- ase reaction and has established roles for specific residues in all steps of catalysis from substrate binding to product release. However, there is still muchthatcanbelearnedfromtheseenzymes.Ofparticularinterestarejust how product release is controlled and the role of the incoming isoprenoid substrate in this process, and the unresolved differences in the catalytic mechanisms of FTase and GGTase-I including the stabilization of the diphosphate leaving group. In addition, it will be important to determine justhowmanyCAAXproteinsinmammaliancellsareactuallyprocessedby theseenzymes,asultimatelytheseproteinsarethearbitersofthebiological impactoftheseenzymes.Recentadvancesonthechemistryfront,including caged isoprenoid diphosphate substrates [63], and those with affinity tags compatible with enzyme recognition [64,65], should facilitate these and otherimportantstudiesontheCAAXprenyltransferases. ACKNOWLEDGMENTS Workintheauthors’laboratorywassupportedbytheNIHandAmericanCancerSociety. REFERENCES 1. Zhang, F.L., and Casey, P.J. (1996). Protein prenylation: molecular mechanisms and functionalconsequences.AnnuRevBiochem65:241–269. 2. Gelb,M.H.,Brunsveld,L.,Hrycyna,C.A.,Michaelis,S.,Tamanoi,F.,VanVoorhis,W.C., andWaldmann,H.(2006).Therapeuticinterventionbasedonproteinprenylationand associatedmodifications.NatChemBiol2:518–528.