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.

Six degrees head-down tilt bed rest caused low-grade hemolysis: a prospective randomized clinical trial

“…Aborting circadian oscillations may alter erythrocyte control since both heme synthesis and degradation molecules are central actors in multiple internal clocks and metabolic pathways 64,65 . Heme binding to CLOCK protein regulates circadian control 66 . Both heme biosynthesis enzyme delta-aminolevulinate synthase 1 and hemolysis enzymes HO in liver followed a circadian expression [67][68][69] .…”

Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel

“…Despite attempts to obtain a co-crystal structure of heme-bound hERG3-eag, extensive co-crystallization and soaking experiments were unsuccessful. As we have noted previously (6), this does not mean that heme cannot bind to the protein, because the spectroscopic data clearly demonstrate that it does. It is more likely that the particular conformation selected for in the crystal structure is incompatible with heme binding, so that heme cannot bind to the protein during the crystallization process.…”

“…The FT analyzer was set to acquire 10 microscans over the m/z range 800-2000 Da in positive ion mode. The maximum injection time for MS was 150 ms, and the AGC target setting was 3e 6 . Protein chargestate distributions were deconvoluted using the Xtract function of the Xcalibur program (version 2.1.0.1139, Thermo Scientific).…”

“…Several layers of regulatory control can be envisaged in the process of heme binding to a PAS domain protein. Heme binding might, for example, induce a conformational change in the PAS domain, which affects the interactions with partner proteins, and there is evidence for this in the heme-binding circadian proteins (6). Because known cell-signaling gases such as CO, NO, and O 2 all bind to heme (and with different affinities), the formation of a heme-bound PAS domain opens opportunities for the molecule to also respond to changes in concentrations of any or all of these cell-signaling gases.…”

An Analysis of the Multifaceted Roles of Heme in the Pathogenesis of Cancer and Related Diseases

“…In fact, several epidemiological studies have linked circadian disruption, for example, in the case of shift work, to increased occurrence of cancers [92][93][94][95][96][97][98][99][100][101], and the IARC lists nightshift work, which disrupts CR as a carcinogen [97,102]. The Circadian Clock is regulated via circadian locomotor output cycles kaput (CLOCK) and Brain and muscle ARNT-Like 1 (BMAL 1), which are basic helix-loop-helix PER-ARNT-SIM transcription factors [103]. Nearly all tumor types exhibit alteration in the expression of CLOCK genes [104].…”