Heme binding to human CLOCK affects interactions with the E-box Samuel L. Freemana, Hanna Kwona, Nicola Portolanob,c, Gary Parkinb,c, Umakhanth Venkatraman Girijab,c,1, Jaswir Basranb,c, Alistair J. Fieldingd, Louise Fairallc,e, Dimitri A. Svistunenkof, Peter C. E. Moodyc,e, John W. R. Schwabec,e, Charalambos P. Kyriacoug, and Emma L. Ravena,2 aSchool of Chemistry, University of Bristol, BS8 1TS Bristol, United Kingdom; bDepartment of Chemistry, University of Leicester, LE1 7RH Leicester, United Kingdom; cLeicester Institute of Structural and Chemical Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; dSchool of Pharmacy and Biomolecular Science, Liverpool John Moores University, Liverpool L3 3AF, United Kingdom; eDepartment of Molecular and Cell Biology, University of Leicester, LE1 7RH Leicester, United Kingdom; fSchool of Biological Sciences, University of Essex, Colchester, Essex CO4 3SQ, United Kingdom; and gDepartment of Genetics and Genome Biology, University of Leicester, LE1 7RH Leicester, United Kingdom Edited by John T. Groves, Princeton University, Princeton, NJ, and approved August 12, 2019 (received for review March 28, 2019) The circadian clock is an endogenous time-keeping system that is ligands—such as oxygen (O2), carbon monoxide (CO), and nitric ubiquitous in animals and plants as well as some bacteria. In oxide (NO)—which are themselves established as important mammals, the clock regulates the sleep–wake cycle via 2 basic signaling molecules in cellular control (4). helix–loop–helix PER-ARNT-SIM (bHLH-PAS) domain proteins— The 2 E-box binding proteins—NPAS2 and CLOCK—belong CLOCK and BMAL1. There is emerging evidence to suggest that to the family of transcriptional regulators that contain both basic – – heme affects circadian control, through binding of heme to various helix loop helix (bHLH) domains at the N terminus as well as 2 circadian proteins, but the mechanisms of regulation are largely PAS domains. High sequence identity between these 2 proteins unknown. In this work we examine the interaction of heme with (5, 6) was noted before a direct link between heme and any human CLOCK (hCLOCK). We present a crystal structure for the circadian protein had been established. In this work we present an analysis of heme binding to the PAS-A domain of human PAS-A domain of hCLOCK, and we examine heme binding to the – PAS-A and PAS-B domains. UV-visible and electron paramagnetic CLOCK, we examine the effect of heme on the CLOCK DNA binding interaction, and we present a structure for the PAS-A resonance spectroscopies are consistent with a bis-histidine li- BIOCHEMISTRY domain. We use this information to put forward ideas on how gated heme species in solution in the oxidized (ferric) PAS-A pro- heme binding might be linked to regulation of circadian control. tein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which Results may give rise to an additional Cys axial ligand at 20K (His/Cys Crystal Structure of hCLOCK PAS-A. A recombinant version (N-terminal coordination). Using DNA binding assays, we demonstrate that His tagged) of the PAS-A domain of hCLOCK (hCLOCK PAS-A, heme disrupts binding of CLOCK to its E-box DNA target. Evidence Fig. 2) was expressed in Escherichia coli. The purified protein is presented for a conformationally mobile protein framework, crystallized as a homodimer in the apo-form (i.e., without heme), CHEMISTRY which is linked to changes in heme ligation and which has the consistent with behavior of the protein during size exclusion capacity to affect binding to the E-box. Within the hCLOCK struc- chromatography (SI Appendix,Fig.S2). tural framework, this would provide a mechanism for heme- dependent transcriptional regulation. Significance heme | circadian | CLOCK Heme proteins have several well-established functions in bi- ology, from oxygen transport to electron transfer and catalysis. he circadian clock is the internal timekeeping system that But in addition, there is new evidence that heme has a wider Tgenerates a daily rhythm in physiology, biochemistry, and regulatory role in the cell. This includes a role for heme in the behavior of almost all higher organisms and some prokaryotes. – regulation of circadian rhythm. In this work, we have examined In mammalian systems, as in insects, a transcriptional trans- the binding of heme to human CLOCK, one of the positive el- lational feedback loop appears to be at the core of the oscillator ements in the circadian autoregulatory feedback loop. We find (1), and this is coupled to an evolutionarily more primitive – evidence for a conformational mobility within the heme metabolic oscillator (2). The transcriptional translational feed- pocket, and that heme disrupts binding of CLOCK to its E-box back loop has a number of positive and negative components DNA. These results provide a direct link between heme binding that drive interconnected loops that generate the molecular and DNA partner recognition that may form the basis for a clockworks. In mammals, NPAS2 (in the forebrain) and CLOCK mechanism of regulatory control. (in the suprachiasmatic nucleus) form heterodimers with BMAL1 (brain and muscle arnt-like 1) and represent the positive limb of Author contributions: P.C.E.M., J.W.R.S., C.P.K., and E.L.R. designed research; S.L.F., H.K., the feedback loop (Fig. 1). Both dimers bind to the same E-box N.P., G.P., U.V.G., J.B., A.J.F., L.F., and D.A.S. performed research; S.L.F., H.K., N.P., G.P., DNA sequence (CANNTG) to activate expression of the negative U.V.G., J.B., A.J.F., L.F., D.A.S., P.C.E.M., J.W.R.S., C.P.K., and E.L.R. analyzed data; and autoregulators, the 3 Period (Per) and 2 Cryptochrome (Cry)genes E.L.R. wrote the paper with contribution from all authors. (1). PER and CRY, after various posttranslational modifications The authors declare no conflict of interest. that build delays into the loop, then interact with the BMAL1 This article is a PNAS Direct Submission. complexes to negatively regulate their own genes, thereby closing Published under the PNAS license. the loop (Fig. 1 and ref. 1). REV-ERBα, a member of the nuclear Data deposition: The atomic coordinates and structure factors have been deposited in the receptor family, also functions in the cell to repress tran- Protein Data Bank, www.rcsb.org (PDB ID code 6QPJ). scription of BMAL1. 1Present address: Faculty of Health and Life Sciences, De Montfort University, Leicester There is an accumulating body of evidence that indicates that LE1 9BH, United Kingdom. heme is a regulator of the circadian oscillator (3), but the mech- 2To whom correspondence may be addressed. Email: [email protected]. anisms of regulation are entirely unknown. Conceptually, heme This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. binding provides a simple mechanism for signal transduction because 1073/pnas.1905216116/-/DCSupplemental. regulation by heme can be coupled to its ability to bind other www.pnas.org/cgi/doi/10.1073/pnas.1905216116 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Simplified figure of mammalian regulatory clockworks (52). In the positive loop, NPAS2 (dark blue circle) and CLOCK (ditto) form heterodimers with BMAL1 (light blue) which bind to the E-box to activate expression of PER (black) and CRY (orange). PER and CRY heterodimers then interact with the BMAL1 heterodimers to negatively regulate their own genes, thereby closing the loop (1). Other CLOCK-related genes are expressed, including the nuclear receptors REV-ERBα/β (green) and the retinoid receptor orphan receptor (ROR, pink). ALAS, which controls the synthesis (and hence the concentrations) of heme, is also clock regulated. See introductory text for details. The structure of hCLOCK PAS-A is presented in Fig. 3A. The to the protein and with a 6-coordinate, low-spin heme species. tertiary structure of the overall PAS fold consists of a series of Electron paramagnetic resonance (EPR) spectra are in agree- antiparallel β-sheets forming a hydrophobic core in which a ment with this assignment. The spectrum of heme-bound signaling molecule can bind (7). A number of α-helices are in hCLOCK at 20 K (Fig. 4B) reveal turning points at g1 = 2.93, 1 close proximity which are believed to be responsible for the g2 = 2.28, and g3 ≥ 1.7 typical of a low-spin, ferric (S = /2) heme propagation of structural changes to the rest of the protein or to species (11). These low-spin g-values are consistent with a bis- a partner protein when in a complex. histidine heme coordination. Weak resonances were also ob- eff ∼ eff ∼ The hCLOCK structure has close similarity to that of the O2 served at 4 K at g 6.0 and g 2, which are characteristic of 5 sensor from Rhizobium (FixL, ref. 8) and the heme-regulated high-spin ferric (S = /2) heme (SI Appendix, Fig. S4A). In ad- phosphodiesterase from E. coli (EcDOS) (9, 10), as shown in dition, a shoulder is observed on the central line and attributed = Fig. 3 B and C. A comparison of the heme binding locations in to overlap of a second (minor) low-spin heme species (g1 2.45, = = the 2 structures is informative (SI Appendix, Fig. S3). In the g2 2.28, and g3 1.92). These g-values are similar to those structure of hCLOCK PAS-A, histidine 144, which is implicated previously observed for the isolated PAS-A domain of mouse in heme binding as below, is found on a loop between 2 short PER2 (12).