recombinant hdac3 smrt dad  (BPS Bioscience)

Journal: Nature

Article Title: Structure of HDAC3 bound to corepressor and inositol tetraphosphate

doi: 10.1038/nature10728

Figure Lengend Snippet: a, Interaction of the SMRT-DAD (green ribbon) with the HDAC3 (grey surface). The Ins(1,4,5,6)P 4 at the interface is shown as a raspberry coloured surface. Side chains in the DAD that mediate interaction with HDAC3 and Ins(1,4,5,6)P 4 are shown as sticks. b, Structure of the DAD domain in solution (PDBcode 1XC5) compared with that bound to HDAC3 (helices are individually coloured to facilitate comparison).

Article Snippet: His6-tagged SMRT-DAD was expressed in E. coli strain Rosetta (Novagen) and initial purification was carried out using Nickel NTA agarose (Qiagen) followed by gel filtration chromatography using a Superdex-S200 26/60 column (GE Healthcare) in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate.

Techniques:

Journal: Nature

Article Title: Structure of HDAC3 bound to corepressor and inositol tetraphosphate

doi: 10.1038/nature10728

Figure Lengend Snippet: a, A striking feature in the difference electron density map (Fo-Fc at 3σ,) observed following molecular replacement b, Electron density corresponding to the Ins(1,4,5,6)P 4 ligand following refinement (2Fo-Fc at 2.25σ). c, Electrostatic surface representation of the HDAC3/SMRT-DAD complex. A strikingly basic pocket is located at the HDAC3:SMRT-DAD interface (indicated by a dashed green line). The active site pocket of HDAC3 is indicated by a yellow cross. The Ins(1,4,5,6)P 4 is not shown for clarity. d, Ins(1,4,5,6)P 4 binding in the basic pocket at the HDAC3:SMRT-DAD interface. e, Detailed interactions of Ins(1,4,5,6)P 4 with HDAC3 (blue) and SMRT-DAD (grey).

Article Snippet: His6-tagged SMRT-DAD was expressed in E. coli strain Rosetta (Novagen) and initial purification was carried out using Nickel NTA agarose (Qiagen) followed by gel filtration chromatography using a Superdex-S200 26/60 column (GE Healthcare) in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate.

Techniques: Binding Assay

Journal: Nature

Article Title: Structure of HDAC3 bound to corepressor and inositol tetraphosphate

doi: 10.1038/nature10728

Figure Lengend Snippet: a, Alignments of key regions of class I HDACs from H.sapiens and S.cerevisiae. Residues that mediate interaction with the Ins(1,4,5,6)P 4 and SMRT-DAD are highlighted in blue and red respectively. Other conserved residues are highlighted grey. b, Alignment of SANT domains from known interaction partners for class I HDACs. Residues that mediate interaction with the Ins(1,4,5,6)P 4 and HDAC3 are highlighted in blue and red respectively. Green arrows indicate residues that impair HDAC3 recruitment and activation when mutated to alanine .

Article Snippet: His6-tagged SMRT-DAD was expressed in E. coli strain Rosetta (Novagen) and initial purification was carried out using Nickel NTA agarose (Qiagen) followed by gel filtration chromatography using a Superdex-S200 26/60 column (GE Healthcare) in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate.

Techniques: Activation Assay

Journal: Nature

Article Title: Structure of HDAC3 bound to corepressor and inositol tetraphosphate

doi: 10.1038/nature10728

Figure Lengend Snippet: a, The SMRT-DAD (grey cartoon) and the Ins(1,4,5,6)P 4 bind adjacent to the HDAC3 (charged surface representation) active site. Acetate and a methionine (lysine mimic) are located in the active site. b, Details of the HDAC3 active site. Key residues and loops are labelled – see text for details. c, Pseudo helix H1 and loops L1 and L6 are shown in blue on the surface of HDAC3. These regions are influenced / stabilised by SMRT-DAD and Ins(1,4,5,6)P 4 binding. d, Comparison of the structures of HDAC3 and HDAC8. Regions of significant difference are coloured blue (HDAC3) and red (HDAC8).

Article Snippet: His6-tagged SMRT-DAD was expressed in E. coli strain Rosetta (Novagen) and initial purification was carried out using Nickel NTA agarose (Qiagen) followed by gel filtration chromatography using a Superdex-S200 26/60 column (GE Healthcare) in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate.

Techniques: Binding Assay

Journal: Nature

Article Title: Structure of HDAC3 bound to corepressor and inositol tetraphosphate

doi: 10.1038/nature10728

Figure Lengend Snippet: a, Effect of HDAC3 mutations on deacetylase activity of complexes with SMRT-DAD. Residues are mutated to their equivalents in HDAC8. Loop 1 (L1) mutation is H17C/G21A/K25I. Loop 6 (L6) mutation is R264P/L265M. Mut-All mutant is H17C/G21A/K25I/R264P/L265M/R301A. b, Immunoblots showing that the HDAC3 mutations perturb interaction with the SMRT-DAD. c, Deacetylase assays of HDAC3-FLAG reconstituted with bacterially-expressed SMRT-DAD in the presence of various inositol phosphates. IP4, IP3 and IP6 are Ins(1,4,5,6)P 4 , Ins(1,4,5)P 3 and Ins(1,2,3,4,5,6)P 6 respectively. d , Synthesis pathway for Ins(1,4,5,6)P 4 from PtdIns(3,4,5)P 3 . Yeast Arg82 converts Ins(1,4,5)P 3 to Ins(1,4,5,6)P 4 . In mammals, both IPMK and PTEN are required to make Ins(1,4,5,6)P 4 .

Article Snippet: His6-tagged SMRT-DAD was expressed in E. coli strain Rosetta (Novagen) and initial purification was carried out using Nickel NTA agarose (Qiagen) followed by gel filtration chromatography using a Superdex-S200 26/60 column (GE Healthcare) in buffer containing 50 mM Tris/HCl pH 7.5, 50 mM potassium acetate.

Techniques: Histone Deacetylase Assay, Activity Assay, Mutagenesis, Western Blot