Skip to content

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

Research output: Contribution to journalArticle

  • Sam L Freeman
  • Hanna Kwon
  • Nicola Portolano
  • Gary Parkin
  • Umakhanth Venkatraman Girija
  • Jaswir Basran
  • Alistair J. Fielding
  • Louise Fairall
  • Dimitri A Svistuneko
  • Peter C. E. Moody
  • John W. R. Schwabe
  • Charalambos P. Kyriacou
  • Emma Raven
Original languageEnglish
JournalProceedings of the National Academy of Sciences of the United States of America
Early online date1 Oct 2019
DOIs
DateAccepted/In press - 29 Aug 2019
DateE-pub ahead of print (current) - 1 Oct 2019

Abstract

The 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 two basic helix-loophelix 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 EPR spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidised (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/Cyscoordination). 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.

    Research areas

  • Heme, circadian

Documents

Documents

  • Full-text PDF (accepted author manuscript)

    Rights statement: This is the accepted author manuscript (AAM). The final published version (version of record) is available online via PNAS at https://doi.org/10.1073/pnas.1905216116 . Please refer to any applicable terms of use of the publisher.

    Accepted author manuscript, 14 MB, PDF document

    Embargo ends: 1/04/20

    Request copy

DOI

View research connections

Related faculties, schools or groups