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Nat Commun
2017 Nov 15;81:1513. doi: 10.1038/s41467-017-01701-2.
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Crystal structures of the mitochondrial deacylase Sirtuin 4 reveal isoform-specific acyl recognition and regulation features.
Pannek M
,
Simic Z
,
Fuszard M
,
Meleshin M
,
Rotili D
,
Mai A
,
Schutkowski M
,
Steegborn C
.
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Sirtuins are evolutionary conserved NAD+-dependent protein lysine deacylases. The seven human isoforms, Sirt1-7, regulate metabolism and stress responses and are considered therapeutic targets for aging-related diseases. Sirt4 locates to mitochondria and regulates fatty acid metabolism and apoptosis. In contrast to the mitochondrial deacetylase Sirt3 and desuccinylase Sirt5, no prominent deacylase activity and structural information are available for Sirt4. Here we describe acyl substrates and crystal structures for Sirt4. The enzyme shows isoform-specific acyl selectivity, with significant activity against hydroxymethylglutarylation. Crystal structures of Sirt4 from Xenopus tropicalis reveal a particular acyl binding site with an additional access channel, rationalizing its activities. The structures further identify a conserved, isoform-specific Sirt4 loop that folds into the active site to potentially regulate catalysis. Using these results, we further establish efficient Sirt4 activity assays, an unusual Sirt4 regulation by NADH, and Sirt4 effects of pharmacological modulators.
Fig. 1. Sirt4 deacylation activities. a Chemical structures of CPS1 peptide and Lys acylations; from top: acetylation, butyrylation, DMS-ylation, HMG-ylation. For the complete set of acyl modifications see Supplementary Fig. 1a. b Sirt4-dependent deacylation of differently acylated CPS1 peptides. (n = 2; error bars: s.d.). c Sirt4 titrations with CPS1 substrate peptide carrying an acetyl, lipoyl, HMG, or DMS modification, respectively. (n = 2; error bars: s.d.). d Comparison of Sirt3, 4, and 5 deacylation activities against substrate peptide with acetyl, succinyl, DMS, or HMG modification, respectively. (n = 2; error bars: s.d.). e Intact protein mass spectrometry of HMG-ylated CypA (unmodified molecular weight 18,012 Da). f Sirt4-dependent deacylation reactions with increasing amounts of untreated and HMG-ylated CypA protein, respectively, as a substrate. (n = 2; error bars: s.d.). g Comparison of the acyl selectivities of Sirt4 from human (hSirt4), clawed frog (xSirt4), and zebrafish (zSirt4) using CPS1 peptide substrates featuring an acetyl, succinyl, DMS, itaconyl, HMG, or lipoyl modification, respectively. (n = 2; error bars: s.d.)
Fig. 2. Crystal structure of xSirt4. a Overall structure of the xSirt4/ADPr complex, with Rossmann-fold domain (green), Zn2+-binding domain (cyan), and a Sirt4-specific loop (blue) indicated. ADPr is shown as sticks, colored according to atom type. Secondary structure elements are numbered equivalent to other sirtuins, elements missing in xSirt4 are indicated by brackets. b xSirt4 active site, with conserved sirtuin catalytic residues and key residues of the Sirt4-loop shown as sticks. ADPr sticks are colored according to atom type and overlaid with 2Fo–Fc electron density (1σ). c Alignment of the Sirt4-loop region in Sirt4 sequences from various chordates (Full alignment: Supplementary Fig. 2c). d Section of a structure-based alignment of Sirt1–6, extended by chordate Sirt1–7 sequences, showing the isoform differences in the Sirt4-loop region (Full alignment: Supplementary Fig. 2d). e Overlay of xSirt4 (gray, blue) with Sirt3 (light gray, red; PDB ID 4BVH) and 5 (dark gray, orange; 4G1C) showing the extended Sirt4-loop, the shorter Sirt5 surface loop, and the short turn in Sirt1–3 (represented by Sirt3)
Fig. 3. Sirt4 structural features and phylogeny. a xSirt4 active site with the additional Sirt4 channel to the acyl pocket shown as transparent surface. ADPr and residues forming the channel are shown as sticks colored according to atom type. Glutaryl-Lys (beige) from an overlaid Sirt5 complex (PDB ID 4UTR) indicates the conventional acyl pocket, and the modeled lipoyl-Lys (cyan) the bottom of the Sirt4 channel. b xSirt4 surface colored according to sequence conservation within Sirt4 isoforms (left; from higher eukaryotic Sirt4 in UniProt) and within the complete Sirtuin family Sirt1–7 (right; from chordate Sirt1–7 in UniProt). Purple indicates high conservation, cyan high variability. c Phylogenetic tree generated from a structure-based sirtuin alignment, extended by aligning 195 chordate sirtuin sequences (see Supplementary Fig. 2c for a core alignment). d Active site inner surface of Sirt4 and Sirt2 (PDB ID 5D7O; see Supplementary Fig. 3d for all isoforms) colored according to electrostatic potential (red: −15 to blue:+15 k
B
T/e). The succinyl- and acetyl-peptide are from overlays (PDB IDs 3RIY and 3GLR, respectively). e Crystal structure of zSirt5 in complex with HMG-CPS1 substrate peptide. Ligand and interacting residues are shown as sticks, and 2Fo–Fc electron density for the peptide is contoured at 1.0σ. Dotted lines indicate hydrogen bonds. f Active site overlay of xSirt4/ADPr (gray) and zSirt5/HMG-CPS1 (blue). Catalytic His and residues analyzed for acyl recognition contributions are shown as sticks and labeled (italics: zSirt5)
Fig. 4. Sirt4 modulation by physiological metabolites and pharmacological compounds. a Effects of known sirtuin modulators on Sirt4 de-HMG-ylation activity. SRT1720 caused effects in controls, indicating incompatibility with the coupled enzymatic assay. (n = 2; error bars: s.d.). b C-site regions of xSirt4 (gray) and Sirt3/Ex527 (gold; PDB ID 4BVB), showing that Ex-527 would clash with the Sirt4-loop residue Pro200. Dotted lines: conserved hydrogen bonds for carbamide recognition. c Effects of SRT1720 and suramin on Sirt4 activity in a HMG-FdL assay. (n = 2; error bars: s.d.). d NAM titration of Sirt4 activity in a HMG-FdL assay. (n = 2; error bars: s.d.). e NADH titration of Sirt4 activity in a HMG-FdL assay (see Supplementary Fig. 4b for a titration in a MS assay). (n = 2; error bars: s.d.)
Ahuja,
Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase.
2007, Pubmed
Ahuja,
Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase.
2007,
Pubmed
Anderson,
SIRT4 Is a Lysine Deacylase that Controls Leucine Metabolism and Insulin Secretion.
2017,
Pubmed
Ashkenazy,
ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules.
2016,
Pubmed
Baur,
Are sirtuins viable targets for improving healthspan and lifespan?
2012,
Pubmed
Choudhary,
The growing landscape of lysine acetylation links metabolism and cell signalling.
2014,
Pubmed
Du,
Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.
2011,
Pubmed
Du,
Investigating the ADP-ribosyltransferase activity of sirtuins with NAD analogues and 32P-NAD.
2009,
Pubmed
Emanuelsson,
Locating proteins in the cell using TargetP, SignalP and related tools.
2007,
Pubmed
Emsley,
Coot: model-building tools for molecular graphics.
2004,
Pubmed
Feldman,
Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins.
2013,
Pubmed
Feldman,
Kinetic and Structural Basis for Acyl-Group Selectivity and NAD(+) Dependence in Sirtuin-Catalyzed Deacylation.
2015,
Pubmed
Fischer,
Sirt5 deacylation activities show differential sensitivities to nicotinamide inhibition.
2012,
Pubmed
Frye,
Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.
2000,
Pubmed
Gai,
Crystal structures of SIRT3 reveal that the α2-α3 loop and α3-helix affect the interaction with long-chain acyl lysine.
2016,
Pubmed
Gertz,
Function and regulation of the mitochondrial sirtuin isoform Sirt5 in Mammalia.
2010,
Pubmed
Gertz,
Ex-527 inhibits Sirtuins by exploiting their unique NAD+-dependent deacetylation mechanism.
2013,
Pubmed
Gertz,
Using mitochondrial sirtuins as drug targets: disease implications and available compounds.
2016,
Pubmed
Gille,
STRAP: editor for STRuctural Alignments of Proteins.
2001,
Pubmed
Haigis,
SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells.
2006,
Pubmed
Heltweg,
Nonisotopic substrate for assaying both human zinc and NAD+-dependent histone deacetylases.
2003,
Pubmed
Ho,
SIRT4 regulates ATP homeostasis and mediates a retrograde signaling via AMPK.
2013,
Pubmed
Jeong,
SIRT4 has tumor-suppressive activity and regulates the cellular metabolic response to DNA damage by inhibiting mitochondrial glutamine metabolism.
2013,
Pubmed
Jiang,
SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine.
2013,
Pubmed
Jin,
SIRT2 Reverses 4-Oxononanoyl Lysine Modification on Histones.
2016,
Pubmed
Jin,
Crystal structures of human SIRT3 displaying substrate-induced conformational changes.
2009,
Pubmed
Kabsch,
XDS.
2010,
Pubmed
Lakshminarasimhan,
Sirt1 activation by resveratrol is substrate sequence-selective.
2013,
Pubmed
Laurent,
SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase.
2013,
Pubmed
Lin,
Protein lysine acylation and cysteine succination by intermediates of energy metabolism.
2012,
Pubmed
Madsen,
Investigating the Sensitivity of NAD+-dependent Sirtuin Deacylation Activities to NADH.
2016,
Pubmed
Mathias,
Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity.
2014,
Pubmed
Maurer,
Inhibitors of the NAD(+)-Dependent Protein Desuccinylase and Demalonylase Sirt5.
2012,
Pubmed
McCoy,
Phaser crystallographic software.
2007,
Pubmed
Michan,
Sirtuins in mammals: insights into their biological function.
2007,
Pubmed
Michishita,
Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins.
2005,
Pubmed
Milne,
Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes.
2007,
Pubmed
Moniot,
Crystal structure analysis of human Sirt2 and its ADP-ribose complex.
2013,
Pubmed
Moniot,
Structures, substrates, and regulators of Mammalian sirtuins - opportunities and challenges for drug development.
2012,
Pubmed
Murshudov,
Refinement of macromolecular structures by the maximum-likelihood method.
1997,
Pubmed
Peck,
SIRT inhibitors induce cell death and p53 acetylation through targeting both SIRT1 and SIRT2.
2010,
Pubmed
Rack,
Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.
2015,
Pubmed
Rauh,
An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms.
2013,
Pubmed
Ringel,
Alternate deacylating specificities of the archaeal sirtuins Sir2Af1 and Sir2Af2.
2014,
Pubmed
Roessler,
Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.
2014,
Pubmed
Sanders,
Structural basis for sirtuin function: what we know and what we don't.
2010,
Pubmed
Sauve,
Sirtuin chemical mechanisms.
2010,
Pubmed
Sauve,
The biochemistry of sirtuins.
2006,
Pubmed
Schmidt,
Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation.
2004,
Pubmed
Schuetz,
Structural basis of inhibition of the human NAD+-dependent deacetylase SIRT5 by suramin.
2007,
Pubmed
Schutkowski,
New assays and approaches for discovery and design of Sirtuin modulators.
2014,
Pubmed
Simic,
The ɛ-Amino Group of Protein Lysine Residues Is Highly Susceptible to Nonenzymatic Acylation by Several Physiological Acyl-CoA Thioesters.
2015,
Pubmed
Smith,
A continuous microplate assay for sirtuins and nicotinamide-producing enzymes.
2009,
Pubmed
Suenkel,
Inhibition of the human deacylase Sirtuin 5 by the indole GW5074.
2013,
Pubmed
Tan,
Lysine glutarylation is a protein posttranslational modification regulated by SIRT5.
2014,
Pubmed
Teng,
Efficient demyristoylase activity of SIRT2 revealed by kinetic and structural studies.
2015,
Pubmed
Tennen,
Functional dissection of SIRT6: identification of domains that regulate histone deacetylase activity and chromatin localization.
2010,
Pubmed
Wagner,
Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix.
2013,
Pubmed
Wagner,
A Class of Reactive Acyl-CoA Species Reveals the Non-enzymatic Origins of Protein Acylation.
2017,
Pubmed
Wagner,
Nonenzymatic protein acylation as a carbon stress regulated by sirtuin deacylases.
2014,
Pubmed
Yang,
NAD metabolism and sirtuins: metabolic regulation of protein deacetylation in stress and toxicity.
2006,
Pubmed
You,
Structural Basis of Sirtuin 6 Activation by Synthetic Small Molecules.
2017,
Pubmed
Zhou,
The bicyclic intermediate structure provides insights into the desuccinylation mechanism of human sirtuin 5 (SIRT5).
2012,
Pubmed
de Oliveira,
The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease.
2017,
Pubmed