Histone deacetylases (HDACs) are protein deacetylases that play a role in repression of gene transcription and are emerging targets in cancer therapy. Here, we characterize the structure and enzymatic activity of the catalytic domain of human HDAC7 (cdHDAC7). Although HDAC7 normally exists as part of a multiprotein complex, we show that cdHDAC7 has a low level of deacetylase activity which can be inhibited by known HDAC inhibitors. The crystal structures of human cdHDAC7 and its complexes with two hydroxamate inhibitors are the first structures of the catalytic domain of class IIa HDACs and demonstrate significant differences with previously reported class I and class IIb-like HDAC structures. We show that cdHDAC7 has an additional class IIa HDAC-specific zinc binding motif adjacent to the active site which is likely to participate in substrate recognition and protein-protein interaction and may provide a site for modulation of activity. Furthermore, a different active site topology results in modified catalytic properties and in an enlarged active site pocket. Our studies provide mechanistic insights into class IIa HDACs and facilitate the design of specific modulators.The level of histone acetylation is regulated by the action of two classes of enzymes, histone acetyltransferases and histone deacetylases (HDACs).3 Histone acetyltransferases and HDACs are found in large multiprotein complexes, and recruitment of histone acetylase or deacetylase complexes by coactivators or corepressors is thought to cause a local change in the chromatin structure, resulting in either activation or repression of gene transcription (1). Humans have 18 HDACs and, based on their sequence similarity to yeast factors, they are grouped into four classes (class I-IV). Class II HDACs are homologous to yeast histone deacetylase HDA1 and have been implicated as global regulators of gene expression during cell differentiation and development (2). In humans, class II HDACs are subdivided into classes IIa (HDAC4, HDAC5, HDAC7, and HDAC9) and IIb (HDAC6 and HDAC10). Class IIa HDACs contain two functionally important regions, a highly conserved C-terminal catalytic domain and an N-terminal extension that has no similarity with other proteins, mediates the signal-dependent shuttling between the nucleus and the cytoplasm, and harbors binding sites for transcriptional regulators (2, 3). Class IIa HDACs interact with corepressors such as N-CoR (nuclear receptor corepressor) and the MEF2 (myocyte enhancer factor 2) family of transcription factors that is not only important for controlling gene expression in normal cellular programs like muscle differentiation, T-cell apoptosis, neuronal survival, and synaptic differentiation but has also been linked to cardiac hypertrophy, asthma, atherosclerosis, hypertension, and other pathological conditions (3-5). To date all four class IIa HDACs have been knocked out in mice, and the resulting abnormal phenotypes have been extensively characterized (6 -9). HDAC7 for example, plays an important role in cardiova...
Sirtuins are NAD(+)-dependent protein deacetylases and are emerging as molecular targets for the development of pharmaceuticals to treat human metabolic and neurological diseases and cancer. To date, several sirtuin inhibitors and activators have been identified, but the structural mechanisms of how these compounds modulate sirtuin activity have not yet been determined. We identified suramin as a compound that binds to human SIRT5 and showed that it inhibits SIRT5 NAD(+)-dependent deacetylase activity with an IC(50) value of 22 microM. To provide insights into how sirtuin function is altered by inhibitors, we determined two crystal structures of SIRT5, one in complex with ADP-ribose, the other bound to suramin. Our structural studies provide a view of a synthetic inhibitory compound in a sirtuin active site revealing that suramin binds into the NAD(+), the product, and the substrate-binding site. Finally, our structures may enable the rational design of more potent inhibitors.
Heme is a ligand for the human nuclear receptors (NR) REV-ERBα and REV-ERBβ, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 Å crystal structure of the REV-ERBβ LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBβ complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.
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