Fellowship Christina Hines: Substrate and Inhibitor Specificity in Neuropeptidases

SUMMARYOFACTIVITIES(Donotexceed3pages.)A.CHANGESSincesubmissionofthelastapplication/progressreport,haveanysignificantchangesoccurredinthetrainingprogram,particularlytheresearchproject,academicstatus,ortimedistributionofactivities(Le.,percentageoftimedevotedtoresearchproject,coursework,teaching,etc.)?Ifso,explain.B.PROGRESSDescribeconciselytheresearchperformedandresearchtrainingobtainedduringthepastyear.Listallcoursesandpublications.CompletetheGenderandMinorityInclusiontable(s)(seebelow),ifapplicable.C.RESEARCHTRAININGPLANSDescribeconciselytheresearchandresearchtrainingplannedfortherequestedbudgetperiod,includinganycoursework.13.A.SUMMARYOFACTIVITIES:CHANGESSincesubmissionofthelastprogressreport,graduateworkhasbeencompletedwiththePh.D.awardedinAugustof2003.MedicalstudieshaveresumedwiththethirdyearmedicalschoolcurriculumbeginninginSeptemberof2003.AfourthyearBiochemistryresearchelectivewasplacedatthebeginningoftheschoolyeartoenablecompletionofgraduatestudiespriortobeginningtherequiredthirdyearcurriculum.SinceOctober2003,timehasbeendistributedasstatedintheoriginalapplication,withbulkofactivitiescenteringonclinicalexperienceandmedicalclasses.About5%oftimeisstilldevotedtoresearch,intheformofapplyingtoandattendingconferencesandparticipationaton-campusM.D./Ph.D.meetings. 13.B.SUMMARYOFACTIVITIES:PROGRESSThePh.D.dissertationhasbeencompleted,boundandsubmittedtothegraduateschoolsincethelastprogressreport.ThedissertationcontainsproprietarydataandisbeingheldthereuntilAugust2004.2-3firstauthormanuscriptsareinpreparation.Asecondauthormanuscriptonthestructureofneurolysin'sclosecousin,thimetoligopeptidase,hasbeenacceptedforpublicationbyJ.BioIChern.Thedissertationtitleis"CrystallographicandFunctionalStudiesontheNeuropeptidaseNeurolysin,"anditinvestigatesdetailsoftheneurolysincatalyticcycleandconditionsfordeterminationofcomplexstructures.Neurolysinexhibitsunusualsubstraterecognitionproperties,selectivelyhydrolyzingcertainpeptidesequenceswithoutapparentsequenceconservation.Neurolysinisalsoimplicatedinpsychoticandpaindisorders,makingtheenzymeanattractivedrugtarget.Experimentswerethereforedesignedtobetterunderstandneurolysinonamolecularlevelinordertofacilitatedesignofhighlyspecificandnovelinhibitors.Detailsofneurolysin'scatalyticcyclewereinvestigatedusingactivesitemutantcrystalstructuresandactivityprofiles.Basedoncomparisonwiththermolysin,theprototypicalzincmetallopeptidase,weknewthatanactivesiteglutamatewascrucialtothepeptidehydrolysismechanism.However,wedidnotknowdetailsofthepreciseroleofthatglutamate.Sinceinhibitorsofzincmetallopeptidasestodateinvolveactivesiteelements,wewantedtobetterunderstandtheroleoftheglutamatealsoforfuturedrugdesignefforts.CrystalstructuresoftheactivesitemutantsE475A,E475LandE475Yindicatethatthezinc,nottheactivesiteglutamateresidue,isresponsibleforgeneral 13B.SUMMARYOFACTIVITIES:PROGRESS(continued)occupancyofthecatalyticwaterinthefirstshellofligands.Animportantfunctionoftheglutamateresidueappeartobeincontrollingthenumberofwaterligandsandcorrectlypositioningthecatalyticwater.Inaddition,theglutamatemayservetocorrectlyorientalonepairofthewaterfornucleophilicattack.KineticmeasurementsshowallE475mutantshaveseverelyreducedactivity,suggestingthatpositioningandpolarizationofwaterareindispensableforenzymeactivityinadditiontotheglutamate'sroleasaprotonshuttle.Insupportofpreviousstudiesbyothers,substratebindingappearedunaffectedbymutationsatthecatalyticglutamate,consistentwiththeconservationofactivesiteconformationinthecrystalstructures.ThecrystalstructureofE475Yafterremovalofthezinccofactorshowsanintactactivesitearchitecture,withthezinc-chelatingresiduesintheirholoenzymeconformations.Thelackofdisorderintheabsenceofzincminimizestheentropiccostofzincbindingandthereforeincreasesaffinityforthemetalion.Magnesiumionspresentinthecrystallizationbufferdidnotbindatthezincsite,indicatingastrongdiscriminationamongdivalentcations.Thisdiscriminationmaybebasedonthepreferredcoordinationgeometryofthemetalion.Inparticular,neurolysinmayprefermetals,likezinc,thatcoordinate4and5ligands,overmagnesium,whichpreferssix-coordinatedoctohedralgeometry. Thestructureofneurolysincomplexedwiththeleadantipsychoticcompoundneuroprilwasdetermined,andissignificantbothforthedemonstrationofanoveltypeofzincmetallopeptidaseinhibitoraswellastheimplicationsofthisstructureforneurolysin'scatalyticmechanism.Thestructureisthefirstexampleofaneuropeptidaseboundtoaninhibitorthatdoesnotoccludetheactivesite.Theinhibitordoesnotalterthestructureoftheenzyme'scatalyticmachinery,suggestingitinhibitsneurolysinbypreventingconformationalchangesassociatedwiththecatalyticcycle.Giventheevidenceforconformationalchangesaccompanyingsubstratebindinginothermetallopeptidases,itseemsmostlikelythatneuroprilindirectlyaffectssubstratebindingbypreventingoralteringcorrespondingconformationalchangesinneurolysin.Thebeautyofthisclassofinhibitorisitsinherentlyhigherspecificityforneurolysinandthimetoligopeptidaseanditssuitabilityasascaffoldfordesigninginhibitorsthatcrossthebloodbrainbarrier.Smallmoleculesthataffectsimilarconformationalchangesinotherneuropeptidasesmayprovideformorespecific,tight-bindingandbioavailabledrugstargetingthisclassofenzymes.