Crystal Structures of Human SIRT3 Displaying Substrate-induced Conformational Changes

SIRT3 is a major mitochondrial NAD⁺-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD⁺. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD⁺. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.Sirtuins are class III histone deacetylases that couple lysine deacetylation with NAD⁺ hydrolysis and are highly conserved in prokaryotes and eukaryotes (1). Mammals possess seven sirtuins, SIRT1–7, that occupy different subcellular compartments such as the nucleus (SIRT1, -6, -7), cytoplasm (SIRT2), and the mitochondria (SIRT3, -4, and -5) (2). They deacetylate lysines not only on histone substrates (3, 4) but also on non-histone substrates such as p53 tumor suppressor protein (5), Foxo transcription factors (6, 7), PGC-1α (8), α-tubulin (9), acetyl-CoA synthetases (10–12), and glutamate dehydrogenase (13). SIRT4 and SIRT6 have been shown to have ADP-ribosyltransferase activity (14–16). Sirtuins have been reported to play important roles in gene silencing (17), cell cycle regulation (18, 19), metabolism (8, 10–12, 14, 20–22), apoptosis (5, 23, 24), the lifespan-extension effects of calorie restriction (25, 26), and circadian rhythms (27–30). Sirtuins have emerged as therapeutic targets for diseases (31) such as type 2 diabetes (32), neurodegenerative diseases (33, 34), inflammation (35), and cancers (36, 37).Several crystal structures of sirtuins have been reported from Thermotoga maritima (Tm) (38–40), Archaeoglobus fulgidus (Af1 and Af2) (38, 41–44), Escherichia coli (45), yeast (Hst2) (46–49), human SIRT2 (50), and SIRT5 (51). Sirtuins contain a conserved enzymatic core with two domains; that is, a large Rossmann fold domain that binds NAD⁺ and a small domain formed by two insertions of the large domain that binds to a zinc atom. The acetylated peptide substrate binds to the cleft between the two domains. Some of the known structures are apo structures with sirtuin protein alone, whereas others are bound to acetylated peptide substrate and/or NAD⁺ and its analogs. These structures revealed the mechanism of action for the deacetylation activity and substrate specificity.SIRT3 localizes in mitochondria (13, 52–54) and is a major mitochondrial deacetylase. Hyperacetylation of mitochondrial proteins have been observed in SIRT3 knock-out mice (13, 55). Several key enzymes involved in energy production in the mitochondria have been identified as SIRT3 substrates. Acetyl-CoA synthetase 2 (AceCS2)² converts acetate into acetyl-CoA in the mitochondria. Deacetylation of AceCS2 at lysine 642 by SIRT3 activates acetyl-CoA synthetase activity, providing increased acetyl-CoA to feed into the tricarboxylic acid cycle (10, 11). Glutamate dehydrogenase converts glutamate to α-ketoglutarate in mitochondria, promoting the metabolism of glutamate and glutamine to be used as fuels for the tricarboxylic acid cycle for ATP production (56). Glutamate dehydrogenase also induces amino acid-stimulated insulin secretion in insulinoma cells (14). Deacetylation of glutamate dehydrogenase by SIRT3 activates its enzyme activity (13, 57). SIRT3 is reported to deacetylate isocitrate dehydrogenase 2 in turn to activate the enzyme (57). Deacetylate isocitrate dehydrogenase 2 oxidatively decarboxylates isocitrate to α-ketoglutarate while converting NAD⁺ to NADH that promotes regeneration of antioxidants. Recently, Ahn et al. (55) reported that SIRT3^−/− mouse embryonic fibroblasts have decreased ATP levels and abnormal activity of Complex 1 of the electron transport chain. Mice lacking SIRT3 showed reduced basal levels of ATP in the heart, kidney, and liver. Therefore, SIRT3 could play an important role in cell metabolism and energy balance. Caloric restriction (CR) extends the lifespan in Caenorhabditis elegans (58), Drosophila (59), yeast (60), and rodents (61) and showed beneficial effects in primates (62, 63) and human subjects (reported by CALORIE Pennington Team). SIRT3 activity is known to be increased by caloric restriction (64). Deacetylate isocitrate dehydrogenase 2 has been shown to be acetylated in the mitochondria of the fed mouse liver and deacetylated in mitochondria of the fasted mouse liver (65). The activity of glutamate dehydrogenase in the liver was also reported to increase by CR (66). Therefore, SIRT3 could be a key player in CR response. Lanza et al. (67) reported that exercise can increase SIRT3 expression level and prevent the decreasing of SIRT3 levels with increasing age. In addition, variability in the SIRT3 gene, which up-regulates SIRT3 expression, is enriched in long-lived individuals (68, 69). SIRT3 mRNA expression level also increased in leptin-treated ob/ob mice that links SIRT3 with the beneficial effects of leptin in regulating body weight and lipid metabolism (70). SIRT3 has also been implicated in selective apoptotic pathways and cell growth control (71) as well as the NAD⁺ salvage pathway that regulates the NAD⁺ level, which is crucial to cell survival (72).Sirtuins have emerged as therapeutic targets to treat many diseases (31). The potential, important roles of SIRT3 in cell metabolism and CR suggest that SIRT3 could be a promising therapeutic target. Our human SIRT3 crystal structures reported here provide molecular information on the conformational changes induced by substrate binding. This is the essential first step for using structural based ligand design for developing SIRT3 modulators.