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. 2015 Jul 2:6:7645.
doi: 10.1038/ncomms8645.

Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

Han Dai  1 , April W Case  2 , Thomas V Riera  2 , Thomas Considine  2 , Jessica E Lee  3 , Yoshitomo Hamuro  3 , Huizhen Zhao  4 , Yong Jiang  4 , Sharon M Sweitzer  4 , Beth Pietrak  4 , Benjamin Schwartz  4 , Charles A Blum  2 , Jeremy S Disch  2 , Richard Caldwell  2 , Bruce Szczepankiewicz  2 , Christopher Oalmann  2 , Pui Yee Ng  2 , Brian H White  2 , Rebecca Casaubon  2 , Radha Narayan  2 , Karsten Koppetsch  2 , Francis Bourbonais  2 , Bo Wu  5 , Junfeng Wang  5 , Dongming Qian  6 , Fan Jiang  6 , Cheney Mao  6 , Minghui Wang  4 , Erding Hu  4 , Joe C Wu  2 , Robert B Perni  2 , George P Vlasuk  2 , James L Ellis  1
Affiliations

Crystallographic structure of a small molecule SIRT1 activator-enzyme complex

Han Dai et al. Nat Commun. .

Abstract

SIRT1, the founding member of the mammalian family of seven NAD(+)-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases.

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Conflict of interest statement

H.D., A.W.C., T.V.R., T.C., H.Z., Y.J., S.M.S., B.P., B.S., C.A.B., J.S.D., R.C., B.S., C.O., P.Y.N., B.H.W., R.C., R.N., K.K., F.B., M.W., E.H., J.C.W., R.B.P., G.P.V. and J.L.E. are employees GlaxoSmithKline. Patent applications relating to the synthetic compounds have been filed.

Figures

Figure 1
Figure 1. Mini-hSIRT1 construct design and characterization.
(a) Schematic diagram of human full-length hSIRT1 and Mini-hSIRT1 constructs. The N-terminal SBD, the central catalytic domain and the CTR are highlighted in red, green and orange. (b) Heat map of the HDX-MS perturbation of binding of 1 to hSIRT1 in the absence or presence of Ac-p53(W5) (Trp-5mer) at six different time points (15–5000, s). (c) Pivot plot of the activation by a chemically diverse STAC set using the Ac-p53(W5) substrate for mini-hSIRT1 versus full-length hSIRT1, as measured by OAcADPR assay. The red line represents y=x correlation. (d) Pivot plot of the STAC activation of mini-hSIRT1(ΔSBD) versus mini-hSIRT1. (e) Pivot plot the STAC activation of mini-hSIRT1(ΔCTR) versus mini-hSIRT1. (f) Pivot plot of the STAC activation of mini-hSIRT1(E230K) versus mini-hSIRT1.
Figure 2
Figure 2. Structure of Mini-hSIRT1/1 complex.
(a) Structure of Mini-hSIRT1/1 complex shown in ribbon diagram. The α-helices and β-strands in the catalytic domain are shown in cyan and orange, respectively. The α-helices in the N-terminal SBD are shown in blue. The β-strands in the C-terminal CTR are shown in red. All the loops are shown in light gray. The STAC 1 is shown in green, red and blue for carbon, oxygen and nitrogen atoms. The zinc ion is shown as a gray sphere. (b) hSIRT1-binding site of 1 with interacting residues shown in stick representation. Hydrogen bonds are shown as yellow dotted lines. (c) Stereo view of the electrostatic surface potential of the N-terminal SBD and electron density map of STAC 1. The electrostatic potential is contoured at the 5 kT/e level, with red denoting negative potential and blue denoting positive potential. The 2Fo-Fc omit map of 1 is contoured at 1.0 σ level. (d) Crystallographic dimer of Mini-hSIRT1/1 complex. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus.
Figure 3
Figure 3. Structures of Mini-hSIRT1-STAC/ligand complex.
(a) Structure of mini-hSIRT1/1/Ac-p53 7-mer/CarbaNAD quaternary complex. The STAC 1 and Ac-p53 7-mer are shown in green, red and blue for carbon, oxygen and nitrogen atoms. The CarbaNAD is shown in cyan, red, blue and orange for carbon, oxygen, nitrogen and phosphate atoms. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus. (b) Structure of mini-hSIRT1/1/2 complex. The mini-hSIRT1 shown in this complex is hSIRT1(183–516)-GS-CTR as the same complex containing hSIRT1(183–505)-(GGGS)2-CTR diffracts to 3.5 Å even though the structures are almost identical. The STAC 1 is shown in green, red and blue for carbon, oxygen and nitrogen atoms. The Inhibitor 2 is shown in cyan, red, blue and yellow for carbon, oxygen, nitrogen and sulfur atoms. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus. (c) Structural comparison of mini-hSIRT1/1 complex (green), mini-hSIRT1/1/Ac-p53 7-mer/CarbaNAD quaternary complex (orange) and mini-hSIRT1/1/2 complex (magenta). (d) Superimposition of the SBD domains from mini-hSIRT1/1 complex (green), mini-hSIRT1/1/Ac-p53 7-mer/CarbaNAD quaternary complex (orange) and mini-hSIRT1/1/2 complex (magenta). (e) Comparison of the STAC-mediated dimer interface of mini-hSIRT1/1 complex (green), mini-hSIRT1/1/Ac-p53 7-mer/CarbaNAD quaternary complex (orange) and mini-hSIRT1/1/2 complex (magenta).
Figure 4
Figure 4. Activation of full-length hSIRT1 mutants.
(a) Heat map of the ratio of wild-type/mutant fold-activation for hSIRT1 mutants for each compound from a structurally diverse collection of 246 STACs. Ratios from 0.80 to 1.16 are colored gray. This range covers one s.d. of the mean for V224A (0.98±0.18-fold, Supplementary Fig. 5b) which does not affect activation. Activation impairment denoted as a red gradient (ratios of 1.17/6.78). Activation enhancement is shown as a blue gradient (ratios of 0.78/0.24). (b) Comparison of the fold-activation of I223R versus wild-type hSIRT1 with a structurally diverse collection of STACs. All of the data were generated with the OAADPr assay using the Ac-p53(W5) substrate. A different compound set (∼250 STACs) was used for the site-directed mutagenesis studies compared with that used for the initial characterization of mini-hSIRT1 (∼80 STACs).
Figure 5
Figure 5. E230K impairs the coupling between STAC and substrate binding and potential role of an electrostatic interaction between Glu230 and Arg446 in the activated conformation.
(a) Heat map of the HDX-MS perturbation of binding of 1 to hSIRT1(E230K) in the absence or presence of Ac-p53(W5) at six different time points (15–5000, s). (b) Pivot plot of the STAC activation of mini-hSIRT1(R446E) versus mini-hSIRT1. (c) Pivot plot of the STAC activation of the double charge-reversal mutant mini-hSIRT1(R446E/E230K) versus mini-hSIRT1. (d) Speculative model of the activated conformation of SIRT1. Glu230 and Arg446 are shown in stick representation. The protein ribbon is rainbow-colored from blue at the N-terminus to red at the C-terminus. 1 and modeled Ac-p53(W5) are shown in cyan and green, respectively.
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References

    1. Finkel T., Deng C. X. & Mostoslavsky R. Recent progress in the biology and physiology of sirtuins. Nature 460, 587–591 (2009). - PMC - PubMed
    1. Baur J. A., Ungvari Z., Minor R. K., Le Couteur D. G. & de Cabo R. Are sirtuins viable targets for improving healthspan and lifespan? Nat. Rev. Drug. Discov. 11, 443–461 (2012). - PMC - PubMed
    1. Frye R. A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem. Biophys. Res. Commun. 273, 793–798 (2000). - PubMed
    1. Haigis M. C. & Sinclair D. A. Mammalian sirtuins: biological insights and disease relevance. Annu. Rev. Pathol. 5, 253–295 (2010). - PMC - PubMed
    1. Verdin E., Hirschey M. D., Finley L. W. & Haigis M. C. Sirtuin regulation of mitochondria: energy production, apoptosis, and signaling. Trends. Biochem. Sci. 35, 669–675 (2010). - PMC - PubMed