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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.)
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