Crystal Structures of Human SIRT3 Displaying Substrate-induced Conformational Changes

Abstract

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 ϩ 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 nonhistone substrates such as p53 tumor suppressor protein (5), Foxo transcription factors (6, 7), PGC-1␣ (8), ␣-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)(28)(29)(30) (50), and SIRT5 (51). Sirtuins contain a conserved enzymatic core with two domains; that is, a large Rossmann fold domain that binds NAD ϩ 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 ϩ 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. Deacetyla...

Ex-527 inhibits Sirtuins by exploiting their unique NAD + -dependent deacetylation mechanism

“…2A) differ only in the cofactor binding loop conformations (see below) from previous structures of these proteins (11)(12)(13)16). In the Sir2Tm/Ex-527 complex, cocrystallized peptide and the ADP ribose part of the soaked-in NAD + are visible.…”

“…Stopping the soak by freezing crystals after 2 min yielded good diffraction data (1.9 Å resolution; Table 1) for a Sir2Tm complex that contained Ex-527 and NAD + derivatives (see below). Human Sirt3, in contrast, readily crystallizes in different states (12,13), and we thus added Ex-527 to the running reaction of Sirt3, NAD + , and acetylated substrate before crystallization. Well-diffracting crystals were obtained (2.0 Å resolution; Table 1) that revealed Ex-527 and a coligand in the NAD + pocket (see below).…”

“…Binding of the protein substrate induces cleft closure (11)(12)(13), and a "cofactor binding loop" gets ordered on NAD + binding (9,14) (Fig. 1A).…”

An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms

“…A homology model for human Sirt1 residues 214-497 (UniProt entry Q96EB6) was generated with Modeller 55 based on an alignment with human Sirt2-34-356 (PDB entry 3ZGO 56 ). Crystal structures of Sirt2 56 , Sirt5 (PDB ID 2B4Y) 57 and Sirt6 (3K35) 58 and the Sirt1 homology model were superimposed on the structure of a Sirt3/peptide complex (3GLR) 59 , and electrostatic potentials were calculated with Adaptive PoissonBoltzmann Solver and mapped on the surface in python molecule viewer 60 .…”

Burning the fat

“…4 DiStefano noted that screening for selective SIRT3 agonists requires "great caution" because previous screens for small molecule sirtuin activators have yielded false positives.…”