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

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 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.

Keywords: CLOCK; circadian; heme.

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.

Fig. 2.

Domain structure in hCLOCK, showing the N-terminal HLH domains and the 2 PAS domains (PAS-A and PAS-B). The protein constructs used in this work are also shown. The vectors used for each construct design are shown in SI Appendix, Fig. S1.

Fig. 3.

A comparison of the structure of PAS-A domains. (A) hCLOCK as presented in this work (PDB code 6QPJ). His144 is indicated in red. This is similar to the equivalent PAS-A domain of mouse CLOCK protein in the CLOCK:BMAL1 complex (53). (B) The O₂ sensor protein from Rhizobium (FixL) (8). (C) The heme-regulated phosphodiesterase from E. coli (EcDOS, refs. and 10). In A, and for mouse CLOCK, the protein crystallizes in the apo-form. In B and C, which both crystallize as the holo-protein, the heme molecule is indicated in red. The Upper figures highlight the binding pocket created by the β-sheets; the Lower structures (which are rotated by 90° about the indicated axis) show the similarity in overall arrangement of the domains. For direct comparison, an overlay of the hCLOCK and FixL structures is presented in SI Appendix, Fig. S3.

Fig. 4.

(A) UV-visible absorption spectra of the ferric heme/CLOCK PAS-A complex. (Inset) Shows ΔAbs at 415 nm (obtained from difference spectra at various heme concentrations) as a function of heme concentration and fitted to a 1:1 binding event. (B) The 9 GHz EPR spectra of ferric heme bound to hCLOCK PAS-A (Top), the H144A variant of PAS-A (Middle), and the C195A variant of PAS-A (Bottom) in 50 mM Tris/HCl buffer at pH 7.0. (C) Absorbance change at 412 nm for the dissociation of heme from hCLOCK PAS-A (3 to 4 μM) on mixing with apo-H64Y/V68F myoglobin (80 μM). Data are fitted to a double exponential process. (D) Spectrum of the ferrous (solid line), CO-bound (dotted line), and NO-bound (dashed line) derivatives of heme-bound hCLOCK PAS-A. (E) Observed rate constants for the association of CO with hCLOCK PAS-A, shown as a function of [CO] (Top) and [hCLOCK PAS-A] (Bottom). (F) Spectrum of the ferric heme/CLOCK PAS-A H144A complex (solid line) compared to free ferric heme (dotted line). This spectrum resembles that of the related His335 variant of NPAS2 PAS-B (where H335 is assigned as 1 of the heme ligands) (21).

Fig. 5.

Electrophoretic mobility shift assays showing DNA binding to the HLH/PAS-A domains of hCLOCK (A and C) and NPAS2 (B and D) in the absence (A and B) and presence (C and D) of heme.

Fig. 6.

The hCLOCK PAS-A crystal structure as presented in this work (in pale blue, PDB code 6QPJ) superimposed onto a homology model of the PAS-A domain of NPAS2 (in light gray). Cys195 (equivalent to Cys171 in NPAS2, overlaid in gray) and Cys194 (equivalent to Tyr169 in NPAS2, not shown) in hCLOCK are shown in yellow; Cys250 in hCLOCK is also shown. His144 in hCLOCK is highlighted in red, overlaid with His119 in NPAS2 (in gray). The homology model was produced using SWISS-MODEL (Biozentrum, University of Basel) using the PAS-A domain of the mouse CLOCK structure (PDB code 4F3L) (54). The figure was created using Pymol (55).