BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR.docVIP

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BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR.doc

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BcL-xL Conformational Changes upon Fragment Binding Revealed by NMR

BcL-xLConformationalChangesuponFragmentBinding RevealedbyNMR Cle′mentineAguirre1,TimtenBrink1,OlivierWalker1,FlorenceGuillie`re1,DanyDavesne2, IsabelleKrimm1* 1UMR5280/Universite′ de Lyon/Universite′ Lyon 1, Institut des Sciences Analytiques, Villeurbanne, France, 2UMR5822/IN2P3/F-69622 Lyon, Universite′ de Lyon, IPNL, Villeurbanne,France Abstract Protein-proteininteractionsrepresentdifficultbutincreasinglyimportanttargetsforthedesignoftherapeuticcompounds able to interfere with biological processes. Recently, fragment-based strategies have been proposed as attractive approachesfortheelaborationofprotein-proteinsurfaceinhibitorsfromfragment-likemolecules.Onemajorchallengein targeting protein-protein interactions is related to the structural adaptation of the protein surface upon molecular recognition. Methods capable of identifying subtle conformational changes of proteins upon fragment binding are thereforerequiredattheearlystepsofthedrugdesignprocess.InthisreportwepresentafastNMRmethodabletoprobe subtleconformationalchangesuponfragmentbinding.Theapproachreliesonthecomparisonofexperimentalfragment- induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering thering-current effect from fragment binding.We illustrate the methodby theretrospective analysis of the complex between the anti-apoptotic Bcl-xL protein and the fragment 49-fluoro-[1,19-biphenyl]-4-carboxylic acid that was previouslyshowntobindoneoftheBcl-xLhotspots.TheCSP-basedapproachshowsthattheproteinundergoesasubtle conformationalrearrangementuponinteraction,forresidueslocatedinhelicesa2,a3andtheverybeginningofa5.Our observations are corroborated by residual dipolar coupling measurements performed on the free and fragment-bound formsoftheBcl-xLprotein.TheseNMR-basedresultsareintotalagreementwithpreviousmoleculardynamiccalculations thatevidencedahighflexibility ofBcl-xL aroundthebindingsite. Hereweshowthat CSPofproteinamineprotons are