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

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.

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

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells.

Histone deacetylases 1 and 2 (HDAC1/2) form the core catalytic components of corepressor complexes that modulate gene expression. In most cell types, deletion of both Hdac1 and Hdac2 is required to generate a discernible phenotype, suggesting their activity is largely redundant. We have therefore generated an ES cell line in which Hdac1 and Hdac2 can be inactivated simultaneously. Loss of HDAC1/2 resulted in a 60% reduction in total HDAC activity and a loss of cell viability. Cell death is dependent upon cell cycle progression, because differentiated, nonproliferating cells retain their viability. Furthermore, we observe increased mitotic defects, chromatin bridges, and micronuclei, suggesting HDAC1/2 are necessary for accurate chromosome segregation. Consistent with a critical role in the regulation of gene expression, microarray analysis of Hdac1/2-deleted cells reveals 1,708 differentially expressed genes. Significantly for the maintenance of stem cell self-renewal, we detected a reduction in the expression of the pluripotent transcription factors, Oct4, Nanog, Esrrb, and Rex1. HDAC1/2 activity is regulated through binding of an inositol tetraphosphate molecule (IP4) sandwiched between the HDAC and its cognate corepressor. This raises the important question of whether IP4 regulates the activity of the complex in cells. By rescuing the viability of double-knockout cells, we demonstrate for the first time (to our knowledge) that mutations that abolish IP4 binding reduce the activity of HDAC1/2 in vivo. Our data indicate that HDAC1/2 have essential and pleiotropic roles in cellular proliferation and regulate stem cell self-renewal by maintaining expression of key pluripotent transcription factors.

Recombinant protein expression for structural biology in HEK 293F suspension cells: a novel and accessible approach.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.