Human sirtuin 5 isoform 1 with suramin

Target ID:

SIRT5

Aliases:

SIR2L5

Deposition Date:

Friday 10th February 2006

Families:

Miscellaneous

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

2NYR

Authors:

Min, J.R., Antoshenko, T., Allali-Hassani, A., Dong, A., Weigelt, J., Sundstrom, M., Arrowsmith, C.H., Edwards, A.M., Bochkarev, A., Plotnikov, A.N., Structural Genomics Consortium (SGC)

Site:

Toronto

ICM Datapack:

ICM Datapack

2NYR

tabs

Structure Details

The NAD+-dependant protein deacetylases play an important role in diverse biological processes such gene silencing, cell cycle progression, chromosome segregation, apoptosis and cell survival. These enzymes, referred to as sirtuins or Sir2 (silent information regulator 2)-like proteins, constitute an ancient family of proteins ranging from bacteria to eukaryotes. Humans have seven proteins of the sirtuin family (SIRT1 through 7) that share a 250 amino acid core domain and have unique specificity, subcellular localization and downstream targets.

Recently, SIRTs have emerged as molecular targets for the development of inhibitors to treat human metabolic and neurological diseases and cancer.

Using forward chemical genetics, several sirtuin inhibitors have been identified. Grozinger et al. screened a 1600-compound library for inhibition on yeast Sir2-mediated silencing at the telomere. Sirtinol, a compound containing a 2-hydroxy-1-napthaldehyde moiety, was the most potent inhibitor, displaying low micromolar IC50 values against ySir2 (68 mM) and SIRT2 (38 mM). Using an analogous strategy, Bedalov et al. discovered a new class of Sir2 inhibitors. These included splitomicin, a compound that diminished gene silencing at all three yeast silent loci and that in vitro, inhibited ySir2 with an IC50 value of 60 mM. Subsequent analysis of 130 splitomicin analogues indicated the requirement for an intact lactone ring. Tervo et al. identified 15 compounds that passed an in silico intestinal absorption test. Examination of these compounds in vitro yielded two that exhibited IC50 values in the low micromolar range.

Recently, we have solved the crystal structure of human SIRT5 in complex with NAD+ at 1.9Å resolution, and now SIRT5 in complex with suramin, which was identified by screening a library of small compounds.

Suramin is (8,8â€™-[carbonylbis [imino-3,1-phenylenecarbonylimino (4-methyl-3,1-phenylene) carbonylimino]] bis-1,3,5-naphtalenetrisulfonic acid, a highly charged polysulfonated compound.

The crystal structure of human SIRT5 in complex with suramin is first structure of SIRT protein with inhibitor bound, and provides information at atomic scale for rational design of new inhibitors.

Materials and Methods

SIRT5 + Suramin

PDB:2NYR

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI: 23408
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated thrombin protease site: MGSSHHHHHHSSGLVPRGS
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gsARPSSSMADFRKFFAKAKHIVIISGAGVSAESGVPTFRGAGGYWRKWQAQDLATPLAFAHNPSRVWEFYHYRREVMGSKEPNAGHRAIAECETRLGKQGRRVVVITQNIDELHRKAGTKNLLEIHGSLFKTRCTSCGVVAENYKSPICPALSGKGAPEPGTQDASIPVEKLPRCEEAGCGGLLRPHVVWFGENLDPAILEEVDRELAHCDLCLVVGTSSVVYPAAMFAPQVAARGVPVAEFNTETTPATNRFRFHFQGPCGTTLPEALA
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:SIRT5 was expressed in E.coli BL21 (DE3) codon plus RIL in TB medium in the presence of 50 µg/ml of kanamycin. Cell were grown at 37oC to an OD600 of 0.8 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15oC.

Purification

Procedure
The crude extract was cleared by centrifugation. The lysate was loaded onto 5 ml HisTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of 30 mM HEPES-NaOH buffer, pH 7.5, containing 500 mM NaCl, 500mM Urea, 50 mM imidazole, 5% glycerol. After that protein was eluted with elution buffer (30 mM HEPES-NaOH buffer, pH 7.5, 500mM Urea, 500mM NaCl, 250 mM imidazole and 5% glycerol). The protein was loaded onto a gel filtration column (Superdex200, 26X60, Amersham Biosciences) equilibrated with buffer 30 mM HEPES-NaOH buffer, pH 7.5, containing 500 mM NaCl at flow rate 4ml/min. The purified protein was treated with thrombin (Sigma) overnight at 4ºC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with 30 mM HEPES-NaOH buffer, pH 7.5 and eluted with linear gradient of NaCl up to 500 mM concentration (30CV). Purification yield was 56 mg of the protein per 1L of culture.

Stock Concentration: 28.5 mg/ml

Enzymatic treatment: Thrombin.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C. For the purification the cell paste was thawed and resuspended in lysis buffer (1xPBS, pH 7.4, 0.25 M NaCl, 5 mM imidazol, 5% glycerol, 0.1% IPTG) with protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:
Ligand
MassSpec:Expected MW is 29386.5, measured mass is 29386.
Crystallization:Purified SIRT5 was complexed with Suramin (Biomol) at 1:5 molar ratio of protein: Suramin, concentrated up to 10 mg/ml and crystallized using hanging drop vapor diffusion method drop at 20°C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 25% PEG 3350, 0.1M Bis-Tris pH 6.5, 0.2M Na Chloride.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Grozinger CM, Chao ED, Blackwell HE, Moazed D, Schreiber SL: Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem 2001, 276:38837-38843.

Bedalov A, Gatbonton T, Irvine WP, Gottschling DE, Simon JA: Identification of a small molecule inhibitor of Sir2p. Proc Natl Acad Sci USA 2001, 98:15113-15118.

Tervo AJ, Kyrylenko S, Niskanen P, Salminen A, Leppanen J, Nyronen TH, Jarvinen T, Poso A: An in silico approach to discovering novel inhibitors of human sirtuin type 2. J Med Chem 2004, 47:6292-6298.