Heme binding to human CLOCK affects interactions with the E-box

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dc.cclicenceN/Aen
dc.contributor.authorFreeman, S.L.
dc.contributor.authorKwon, H.
dc.contributor.authorPortolano, N.
dc.contributor.authorParkin, G.
dc.contributor.authorVenkatraman Girija, U.
dc.contributor.authorBasran, J.
dc.contributor.authorFielding, A.
dc.contributor.authorFairall, L.
dc.contributor.authorSvistunenko, D.
dc.contributor.authorMoody, P.
dc.contributor.authorSchwabe, J.
dc.contributor.authorKyriacou, C.
dc.contributor.authorRaven, E.
dc.date.acceptance2019-08-12
dc.date.accessioned2019-09-26T10:23:58Z
dc.date.available2019-09-26T10:23:58Z
dc.date.issued2019-09-16
dc.descriptionThe file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.en
dc.description.abstractThe circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.en
dc.exception.ref2021codes254aen
dc.funderBBSRC (Biotechnology and Biological Sciences Research Council)en
dc.identifier.citationFreeman, S., Kwon, H., Portolano, N., Parkin, G., Venkatraman Girija, U., Basran, J., Fielding, A., Fairall, L., Svistunenko, D., Moody, P., Schwabe, J., Kyriacou, C. and Raven, E. (2019) Heme binding to human CLOCK affects interactions with the E-box. Proceedings of the National Academy of Sciences, 201905216.en
dc.identifier.doihttps://doi.org/10.1073/pnas.1905216116
dc.identifier.issn0027-8424
dc.identifier.urihttps://dora.dmu.ac.uk/handle/2086/18523
dc.language.isoenen
dc.peerreviewedYesen
dc.projectidgrant BB/L006626/1 to ER (ER = DR EMMA RAVEN, the corresponding author)en
dc.publisherPNASen
dc.researchinstituteInstitute for Allied Health Sciences Researchen
dc.subjectCLOCKen
dc.subjectcircadianen
dc.subjecthemeen
dc.titleHeme binding to human CLOCK affects interactions with the E-boxen
dc.typeArticleen

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