Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

Mariana Sacerdoti; Lissy Gross; Andrew M. Riley; Karin Zehnder; Abhijeet Ghode; Sebastian Klinke; Ganesh Srinivasan Anand; Kristina Paris; Angelika Winkel; Amanda K. Herbrand; H. Yasmin Godage; Gyles Cozier; Evelyn Sub; Jorg O. Schulze; Daniel Pastor-Flores; Mariela Bollini; Maria Victoria Cappellari; Dmitri Svergun; Melissa A. Grawert; Pedro F. Aramendia; Alejandro E. Leroux; Barry Potter; Carlos J. Camacho; Ricardo M. Biondi

doi:10.1126/scisignal.add3184

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

Mariana Sacerdoti, Lissy Gross, Andrew M. Riley, Karin Zehnder, Abhijeet Ghode, Sebastian Klinke, Ganesh Srinivasan Anand, Kristina Paris, Angelika Winkel, Amanda K. Herbrand, H. Yasmin Godage, Gyles Cozier, Evelyn Sub, Jorg O. Schulze, Daniel Pastor-Flores, Mariela Bollini, Maria Victoria Cappellari, Dmitri Svergun, Melissa A. Grawert, Pedro F. AramendiaAlejandro E. Leroux, Barry Potter, Carlos J. Camacho, Ricardo M. Biondi

Department of Life Sciences

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Abstract

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIFpocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by
stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the
selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Original language	English
Article number	eadd3184
Pages (from-to)	eadd3184
Journal	Science Signaling
Volume	16
Issue number	789
DOIs	https://doi.org/10.1126/scisignal.add3184
Publication status	Published - 13 Jun 2023

Bibliographical note

The work was supported by the German Cancer Consortium (DKTK), DFG BI 1044/12-1, 1166 ANPCyT (grants PICT PRH‐2016‐4835; PICT 2016-3525; PICT 2017–0388), and CONICET 1167 to RMB, FOCEM-Mercosur (COF 03/11) to IBioBA and the Wellcome Trust (grants WT082837 1168 to A.M.R. and B.V.L.P. and WT101010 to B.V.L.P.). B.V.L.P. is a Wellcome Trust Senior 1169 Investigator. B.V.L.P. acknowledges an MRC Materials Transfer Agreement with provision of 1170 a PDK1 PH domain construct (residues 408-556) in a pGEX-6P1 vector through the University 1171 of Dundee. SAXS experiments, at the P12 beamline (PETRA III, DESY) of the European 1172 Molecular Biology Laboratory (EMBL) in Hamburg had support from the iNEXT-Discovery 1173 program (project ID: 12843), funded by the Horizon 2020 program of the European 1174 Commission. We are grateful for the access to the PROXIMA2A beamline at Synchrotron 1175 SOLEIL, France (proposal No. 20170984). This research was funded in part, by the Wellcome 1176 Trust. For the purpose of Open Access, the authors have applied a CC BY public copyright 1177 licence to any Author Accepted Manuscript version arising from this submission.

Funding

Acknowledgments: W e thank the institutions that hosted our research and the many colleagueswhoshareddiscussionsandsupportedtheworkov er theyears.W earegratefulto M.E.Guerin,J.O.Cifuente,A.Szalai,andE.Herrmannforvaluableadvice.Funding:Thework wassupportedbytheGermanCancerConsortium(DKTK),DFGBI1044/12-1,ANPCyT(grants PICTPRH-2016-4835,PICT2016-3525,andPICT2017-0388),andCONICETtoR.M.B.;FOCEM-Mercosur(COF03/11)toIBioBA;andtheW ellcome Trust(grantsWT082837toA.M.R.and B.V .L.P .andWT101010toB.V .L.P .). B.V .L.P .isaW ellcome Trus tSeniorInvestigator(grant101010). B.V .L.P . acknowledges an MRC Materials Transfer Agreement with pro vision of a PDK1 PH domainconstruct(residues408–556)inapGEX-6P1vectorthroughtheUniversityofDundee. SAXSexperiments,attheP12beamline(PETRAIII,DESY)oftheEuropeanMolecularBiology Laboratory(EMBL)inHamburg,hadsupportfromtheiNEXT-Discoveryprogram(projectID: 12843),fundedbytheHorizon2020programoftheEuropeanCommission.W earegra teful for theaccesstothePROXIMA-2AbeamlineatSynchrotronSOLEIL,France(proposalno. 20170984).This research was funded, in part, by the W ellcome Trust. Forthe purpose of Open Access,theauthorshaveappliedaCCBYpubliccopyrightlicensetoanyAuthorAccepted Manuscriptversionarisingfromthissubmission.Authorcontributions:Inositol polyphosphatesandderivativ es weredesignedandsynthesizedbyH.Y .G. andA.M.R., supervisedbyB.V .L.P .H.Y .G., A.M.R.,andB.V .L.P .alsoevaluatedactivityonPDK1throughdiverse commercialassaysinapreliminaryfashion.G.E.C.carriedoutlipidoverlaydisplacementassays, supervisedbyA.M.R.andB.V .L.P .A.M.R.andB.V .L.P .conceivedtheoriginalprojecttodevelop inositolphosphatederivativesasinhibitorsofPDK1and,withH.Y .G., broughtupthe paradoxicaleffectsofcompoundsincommercialassaysandpossiblePDK1PHdomain involvementtoR.M.B.R.M.B.supervisedM.S.,K.Z.,E.S.,J.O.S.,D.P .-F ., A.W ., A.H.,L.Z.F .G., and A.E.L.,whoperformedmolecularbiologyandbiochemicalexperimentsandanalyzedresults. Thehigh-qualityproteinsusedinH/Dexchange,crystallography,andSAXSwerepurifiedby L.Z.F .G. underthesupervisionofA.E.L.ThescreeningwasperformedbyM.S.underthe supervisionofR.M.B.andE.S.M.B.supportedtheevaluationofhitsfromthescreening.P .F .A. supervised M.V .C., M.S., and L.Z.F .G. on the validation of results by alternative methods. G.S.A. supervisedA.G.duringtheH/Dexchangeexperiments.SAXSwasperformedbyM.A.G.,andthe results were analyzed by M.A.G., L.Z.F .G., and D.S. S.K. supervised L.Z.F .G. during the crystallographyanalysis.C.J.C.performedmolecularmodelingwithsupportfromK.P .All authorsevaluateddata.R.M.B.ledtheov er allresearchproject.Themanuscriptwaswrittenby R.M.B.withinputfromallcoauthors,whoalsoapprov ed thefinalversion.Competing interests:Theauthorsdeclarethattheydonothavecompetinginterests.Dataandmaterials availability:TherefinedstructureforPDK150–359andvalsartanwasdepositedinthePDBwith theaccessioncode7RLQ.TheSASBDBaccessioncodesareSASDNJ5forSEC-SAXSPDK11–556 andSASDNK5forSEC-SAXSPDK11–556+HYG8.Allotherdataneededtoevaluatethe conclusionsinthepaperarepresentinthepaperortheSupplementaryMaterials.ThepGEX-6P1plasmidexpressingthePDK1PHdomain(residues408–556)isavailablefromD.Alessi underamaterialtransferagreementwithMRCProteinPhosphorylationandUbiquitylation Unit,UK.

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W ellcome Trust

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10.1126/scisignal.add3184

Sacerdoti et alLicence: CC BY

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Sacerdoti, M., Gross, L., Riley, A. M., Zehnder, K., Ghode, A., Klinke, S., Srinivasan Anand, G., Paris, K., Winkel, A., Herbrand, A. K., Godage, H. Y., Cozier, G., Sub, E., Schulze, J. O., Pastor-Flores, D., Bollini, M., Cappellari, M. V., Svergun, D., Grawert, M. A., ... Biondi, R. M. (2023). Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein. Science Signaling, 16(789), eadd3184. Article eadd3184. https://doi.org/10.1126/scisignal.add3184

Sacerdoti, M, Gross, L, Riley, AM, Zehnder, K, Ghode, A, Klinke, S, Srinivasan Anand, G, Paris, K, Winkel, A, Herbrand, AK, Godage, HY, Cozier, G, Sub, E, Schulze, JO, Pastor-Flores, D, Bollini, M, Cappellari, MV, Svergun, D, Grawert, MA, Aramendia, PF, Leroux, AE, Potter, B, Camacho, CJ & Biondi, RM 2023, 'Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein', Science Signaling, vol. 16, no. 789, eadd3184, pp. eadd3184. https://doi.org/10.1126/scisignal.add3184

@article{b444d00d0e5743be849aad9f60f6d8d0,

title = "Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein",

abstract = "The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIFpocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.",

author = "Mariana Sacerdoti and Lissy Gross and Riley, {Andrew M.} and Karin Zehnder and Abhijeet Ghode and Sebastian Klinke and {Srinivasan Anand}, Ganesh and Kristina Paris and Angelika Winkel and Herbrand, {Amanda K.} and Godage, {H. Yasmin} and Gyles Cozier and Evelyn Sub and Schulze, {Jorg O.} and Daniel Pastor-Flores and Mariela Bollini and Cappellari, {Maria Victoria} and Dmitri Svergun and Grawert, {Melissa A.} and Aramendia, {Pedro F.} and Leroux, {Alejandro E.} and Barry Potter and Camacho, {Carlos J.} and Biondi, {Ricardo M.}",

note = "The work was supported by the German Cancer Consortium (DKTK), DFG BI 1044/12-1, 1166 ANPCyT (grants PICT PRH‐2016‐4835; PICT 2016-3525; PICT 2017–0388), and CONICET 1167 to RMB, FOCEM-Mercosur (COF 03/11) to IBioBA and the Wellcome Trust (grants WT082837 1168 to A.M.R. and B.V.L.P. and WT101010 to B.V.L.P.). B.V.L.P. is a Wellcome Trust Senior 1169 Investigator. B.V.L.P. acknowledges an MRC Materials Transfer Agreement with provision of 1170 a PDK1 PH domain construct (residues 408-556) in a pGEX-6P1 vector through the University 1171 of Dundee. SAXS experiments, at the P12 beamline (PETRA III, DESY) of the European 1172 Molecular Biology Laboratory (EMBL) in Hamburg had support from the iNEXT-Discovery 1173 program (project ID: 12843), funded by the Horizon 2020 program of the European 1174 Commission. We are grateful for the access to the PROXIMA2A beamline at Synchrotron 1175 SOLEIL, France (proposal No. 20170984). This research was funded in part, by the Wellcome 1176 Trust. For the purpose of Open Access, the authors have applied a CC BY public copyright 1177 licence to any Author Accepted Manuscript version arising from this submission. ",

year = "2023",

month = jun,

day = "13",

doi = "10.1126/scisignal.add3184",

language = "English",

volume = "16",

pages = "eadd3184",

journal = "Science Signaling",

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number = "789",

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T1 - Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

AU - Sacerdoti, Mariana

AU - Gross, Lissy

AU - Riley, Andrew M.

AU - Zehnder, Karin

AU - Ghode, Abhijeet

AU - Klinke, Sebastian

AU - Srinivasan Anand, Ganesh

AU - Paris, Kristina

AU - Winkel, Angelika

AU - Herbrand, Amanda K.

AU - Godage, H. Yasmin

AU - Cozier, Gyles

AU - Sub, Evelyn

AU - Schulze, Jorg O.

AU - Pastor-Flores, Daniel

AU - Bollini, Mariela

AU - Cappellari, Maria Victoria

AU - Svergun, Dmitri

AU - Grawert, Melissa A.

AU - Aramendia, Pedro F.

AU - Leroux, Alejandro E.

AU - Potter, Barry

AU - Camacho, Carlos J.

AU - Biondi, Ricardo M.

N1 - The work was supported by the German Cancer Consortium (DKTK), DFG BI 1044/12-1, 1166 ANPCyT (grants PICT PRH‐2016‐4835; PICT 2016-3525; PICT 2017–0388), and CONICET 1167 to RMB, FOCEM-Mercosur (COF 03/11) to IBioBA and the Wellcome Trust (grants WT082837 1168 to A.M.R. and B.V.L.P. and WT101010 to B.V.L.P.). B.V.L.P. is a Wellcome Trust Senior 1169 Investigator. B.V.L.P. acknowledges an MRC Materials Transfer Agreement with provision of 1170 a PDK1 PH domain construct (residues 408-556) in a pGEX-6P1 vector through the University 1171 of Dundee. SAXS experiments, at the P12 beamline (PETRA III, DESY) of the European 1172 Molecular Biology Laboratory (EMBL) in Hamburg had support from the iNEXT-Discovery 1173 program (project ID: 12843), funded by the Horizon 2020 program of the European 1174 Commission. We are grateful for the access to the PROXIMA2A beamline at Synchrotron 1175 SOLEIL, France (proposal No. 20170984). This research was funded in part, by the Wellcome 1176 Trust. For the purpose of Open Access, the authors have applied a CC BY public copyright 1177 licence to any Author Accepted Manuscript version arising from this submission.

PY - 2023/6/13

Y1 - 2023/6/13

N2 - The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIFpocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

AB - The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIFpocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

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Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

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