The energetics and ion coupling of cholesterol transport through Patched1

TB Ansell; RA Corey; LV Viti; M Kinnebrew; R Rohatgi; C Siebold; Mark S P Sansom

doi:10.1126/sciadv.adh1609

The energetics and ion coupling of cholesterol transport through Patched1

TB Ansell, RA Corey, LV Viti, M Kinnebrew, R Rohatgi, C Siebold, Mark S P Sansom^*

^*Corresponding author for this work

School of Physiology, Pharmacology & Neuroscience

Research output: Contribution to journal › Article (Academic Journal) › peer-review

3 Citations (Scopus)

Abstract

Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein–coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between “inward-open” and solvent “occluded” states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

Original language	English
Article number	eadh1609
Pages (from-to)	1-15
Journal	Science Advances
Volume	9
Issue number	34
DOIs	https://doi.org/10.1126/sciadv.adh1609
Publication status	Published - 23 Aug 2023

Bibliographical note

Funding Information:
Acknowledgments:W ethankO.VickeryforguidancewithPMFcalculationsanduseofthe pmf.pytoolandW .SongforuseofthePyLipIDtoolkit.Funding:T .B.A., R.A.C.,andM.S.P .S. are supportedbyW ellcome (102164/Z/13/Zand208361/Z/17/Z).ForthepurposeofOpenAccess, theauthorhasappliedaCCBYpubliccopyrightlicensetoanyAuthorAcceptedManuscript version arising from this submission. M.S.P .S. ’s research is additionally supported by the BBSRC (BB/R00126X/1)andPRA CE (PartnershipforAdvancedComputinginEurope;2016163984).C.S. issupportedbytheCancerResearchUK(C20724/A26752)andtheBBSRC(BB/T01508X/1).R.R. issupportedbytheNationalInstitutesofHealth(GM118082andGM106078).M.K.isinreceipt ofapredoctoralfellowshipfromtheNationalScienceFoundation.L.V .V .issupportedbya CancerResearchUKstudentship(C20724/A27829).ThisprojectmadeuseofARCHER supercomputingtimeundertheUKHigh-EndComputingConsortiumforBiomolecular Simulation(HECBioSim;EPSRCEP/R029407/1).Authorcontributions:T .B.A. andR.A.C. performedandanalyzedsimulations.L.V .V .andC.S.clonedandexpressedPTCH1mutants.M.K. andR.R.performedPTCH1functionalassays.M.S.P .S. andC.S.conceptualizedtheproject.T .B.A., R.A.C.,andM.S.P .S. wrotethepaperwithinputfromallauthors.Competinginterests:R.R.and C.S.areconsultantsforDarkBlueTherapeutics.Theauthorsdeclarethattheyhav enoother competinginterests.Dataandmaterialsavailability:Alldataneededtoevaluatethe conclusionsinthepaperarepresentinthepaperand/ortheSupplementaryMaterials.CGfree energyprofilesandatomistictrajectoriesaredeposedonZenodo(DOI:10.5281/zenodo. 7966308).Expressionvectorsandcelllinesusedinthisstudyarefreelyavailablefromthe authors([email protected]@strubi.ox.ac.uk)andtheconstructsequencesand providedwithintheSupplementaryMaterials.

Publisher Copyright:
© 2023 The Authors.

Keywords

Membrane protein
TRANSPORTERS
CHOLESTEROL

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@article{0417a5b55db64184a8a1198db03b1e9d,

title = "The energetics and ion coupling of cholesterol transport through Patched1",

abstract = "Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein–coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between “inward-open” and solvent “occluded” states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.",

keywords = "Membrane protein, TRANSPORTERS, CHOLESTEROL",

author = "TB Ansell and RA Corey and LV Viti and M Kinnebrew and R Rohatgi and C Siebold and Sansom, {Mark S P}",

note = "Funding Information: Acknowledgments:W ethankO.VickeryforguidancewithPMFcalculationsanduseofthe pmf.pytoolandW .SongforuseofthePyLipIDtoolkit.Funding:T .B.A., R.A.C.,andM.S.P .S. are supportedbyW ellcome (102164/Z/13/Zand208361/Z/17/Z).ForthepurposeofOpenAccess, theauthorhasappliedaCCBYpubliccopyrightlicensetoanyAuthorAcceptedManuscript version arising from this submission. M.S.P .S. {\textquoteright}s research is additionally supported by the BBSRC (BB/R00126X/1)andPRA CE (PartnershipforAdvancedComputinginEurope;2016163984).C.S. issupportedbytheCancerResearchUK(C20724/A26752)andtheBBSRC(BB/T01508X/1).R.R. issupportedbytheNationalInstitutesofHealth(GM118082andGM106078).M.K.isinreceipt ofapredoctoralfellowshipfromtheNationalScienceFoundation.L.V .V .issupportedbya CancerResearchUKstudentship(C20724/A27829).ThisprojectmadeuseofARCHER supercomputingtimeundertheUKHigh-EndComputingConsortiumforBiomolecular Simulation(HECBioSim;EPSRCEP/R029407/1).Authorcontributions:T .B.A. andR.A.C. performedandanalyzedsimulations.L.V .V .andC.S.clonedandexpressedPTCH1mutants.M.K. andR.R.performedPTCH1functionalassays.M.S.P .S. andC.S.conceptualizedtheproject.T .B.A., R.A.C.,andM.S.P .S. wrotethepaperwithinputfromallauthors.Competinginterests:R.R.and C.S.areconsultantsforDarkBlueTherapeutics.Theauthorsdeclarethattheyhav enoother competinginterests.Dataandmaterialsavailability:Alldataneededtoevaluatethe conclusionsinthepaperarepresentinthepaperand/ortheSupplementaryMaterials.CGfree energyprofilesandatomistictrajectoriesaredeposedonZenodo(DOI:10.5281/zenodo. 7966308).Expressionvectorsandcelllinesusedinthisstudyarefreelyavailablefromthe authors([email protected]@strubi.ox.ac.uk)andtheconstructsequencesand providedwithintheSupplementaryMaterials. Publisher Copyright: {\textcopyright} 2023 The Authors.",

year = "2023",

month = aug,

day = "23",

doi = "10.1126/sciadv.adh1609",

language = "English",

volume = "9",

pages = "1--15",

journal = "Science Advances",

issn = "2375-2548",

publisher = "American Association for the Advancement of Science",

number = "34",

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TY - JOUR

T1 - The energetics and ion coupling of cholesterol transport through Patched1

AU - Ansell, TB

AU - Corey, RA

AU - Viti, LV

AU - Kinnebrew, M

AU - Rohatgi, R

AU - Siebold, C

AU - Sansom, Mark S P

N1 - Funding Information: Acknowledgments:W ethankO.VickeryforguidancewithPMFcalculationsanduseofthe pmf.pytoolandW .SongforuseofthePyLipIDtoolkit.Funding:T .B.A., R.A.C.,andM.S.P .S. are supportedbyW ellcome (102164/Z/13/Zand208361/Z/17/Z).ForthepurposeofOpenAccess, theauthorhasappliedaCCBYpubliccopyrightlicensetoanyAuthorAcceptedManuscript version arising from this submission. M.S.P .S. ’s research is additionally supported by the BBSRC (BB/R00126X/1)andPRA CE (PartnershipforAdvancedComputinginEurope;2016163984).C.S. issupportedbytheCancerResearchUK(C20724/A26752)andtheBBSRC(BB/T01508X/1).R.R. issupportedbytheNationalInstitutesofHealth(GM118082andGM106078).M.K.isinreceipt ofapredoctoralfellowshipfromtheNationalScienceFoundation.L.V .V .issupportedbya CancerResearchUKstudentship(C20724/A27829).ThisprojectmadeuseofARCHER supercomputingtimeundertheUKHigh-EndComputingConsortiumforBiomolecular Simulation(HECBioSim;EPSRCEP/R029407/1).Authorcontributions:T .B.A. andR.A.C. performedandanalyzedsimulations.L.V .V .andC.S.clonedandexpressedPTCH1mutants.M.K. andR.R.performedPTCH1functionalassays.M.S.P .S. andC.S.conceptualizedtheproject.T .B.A., R.A.C.,andM.S.P .S. wrotethepaperwithinputfromallauthors.Competinginterests:R.R.and C.S.areconsultantsforDarkBlueTherapeutics.Theauthorsdeclarethattheyhav enoother competinginterests.Dataandmaterialsavailability:Alldataneededtoevaluatethe conclusionsinthepaperarepresentinthepaperand/ortheSupplementaryMaterials.CGfree energyprofilesandatomistictrajectoriesaredeposedonZenodo(DOI:10.5281/zenodo. 7966308).Expressionvectorsandcelllinesusedinthisstudyarefreelyavailablefromthe authors([email protected]@strubi.ox.ac.uk)andtheconstructsequencesand providedwithintheSupplementaryMaterials. Publisher Copyright: © 2023 The Authors.

PY - 2023/8/23

Y1 - 2023/8/23

N2 - Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein–coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between “inward-open” and solvent “occluded” states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

AB - Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein–coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between “inward-open” and solvent “occluded” states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

KW - Membrane protein

KW - TRANSPORTERS

KW - CHOLESTEROL

UR - http://europepmc.org/abstract/med/37611095

U2 - 10.1126/sciadv.adh1609

DO - 10.1126/sciadv.adh1609

M3 - Article (Academic Journal)

C2 - 37611095

SN - 2375-2548

VL - 9

SP - 1

EP - 15

JO - Science Advances

JF - Science Advances

IS - 34

M1 - eadh1609

ER -

The energetics and ion coupling of cholesterol transport through Patched1

Abstract

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Keywords

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