Human histone deacetylase 6, Znf ubiquitin binding domain, in complex with F1967-0912 (3-[6-Oxo-3-(3-pyridinyl)-1(6H)-pyridazinyl]propanoic acid)

Target ID:

HDAC6

Aliases:

FLJ16239HD6JM21

Deposition Date:

Tuesday 14th June 2016

Families:

TransferasesUbiquitin Related Biology

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

5KH7

Authors:

Harding RJ, Walker J, Ravichandran M, Schapira M, Bountra C, Edwards AM, Santhakumar V, Arrowsmith CM

Site:

Toronto

5KH7

tabs

Structure Details

HDAC6 is a cytosolic deacetylase which functions in a diverse range of cellular processes including cell trafficking, autophagy and cell signalling. It is composed of two catalytic domains as well as a unique zinc-finger ubiquitin-binding domain (ZnF-UBD). HDAC6 gathers scattered polyubiquitinated protein aggregates via the ZnF-UBD and loads them on dynein/microtubules for aggresome degradation (Kawagushi et al. Cell 2003). Catalytic domain inhibitors antagonize the interaction of HDAC6 with dynein (Hideshima et al. PNAS 2005) and disrupt transport to the aggresome through acetylation of α-tubulin (Amengual et al. Clin. Cancer Res. 2015). Synergistic inhibition of HDAC6 and the proteasome is a promising strategy against myeloma and lymphoma (Santo et al. Blood 2012, Amengual et al. Clin. Cancer Res. 2015). Current selective catalytic HDAC6 inhibitors include hydroxamic acids, and toxicity may be an issue. Targeting the recognition of protein aggregates with ZnF-UBD inhibitors rather than their loading on microtubules (as catalytic inhibitors do) could be a more direct way to antagonize aggresome degradation. SGC Toronto previously solved the apo structure of the ZnF-UBD (PDB code 3C5K) as well as in complex with the C-terminal ubiquitin peptide RLRGG, the native substrate for this domain (PDB code 3GV4). This cocrystal structure is one of the first of this domain with a ligand bound in the ubiquitin binding pocket.

Materials and Methods

HDAC6

PDB:5KH7
Entry Clone Accession:BC013737
Entry Clone Source:MGC AT36-C10
SGC Clone Accession:JMC105-C01
Tag:N-terminal His6-tag, removed
Host:BL21(DE3)V2R-pRARE2
Vector:pET28-lic
Prelude:HDAC6:P1109-H1213The sequence is consistent with reference sequence NP_006035.

Sequence: gsPLPWCPHLVAVCPIPAAGLDVTQPCGDCGTIQENWVCLSCYQVYCGRYINGHMLQHHGNSGHPLVLSYIDLSAWCYYCQAYVHHQALLDVKNIAHQNKFGEDMPHDNA sequence has been verified by sequencing

Growth

Procedure:Shaker - Cultures were grown in M9 media supplemented with 50 µM ZnCl2, and grown at 37 °C until OD600 reached 0.6 before induction with 0.2 mM IPTG and overnight growth at 15 °C.

Purification

Buffers: Washing Buffer: 50 mM tris-pH8, 300 mM NaCl, 25 mM imidazole, 2 mM TCEP - Elution Buffer: 50 mM tris-pH8, 300 mM NaCl, 300 mM imidazole, 2 mM TCEP - Dialysis Buffer: 50 mM tris-pH8, 150 mM NaCl, 2 mM TCEP, 2 mM CaCl2 - Size-exclusion buffer: 50 mM tris-pH8, 150 mM NaCl, 2 mM TCEP.

Procedure: Following sonication, lysates were clarified by centrifugation, 15000 rpm, 1 hour, JLA16.250. Proteins were purified first using Ni-affinity chromatography with NiNTA resin (Qiagen) (1 mL slurry per L cells) washing 100 CV extraction buffer then 100 CV washing buffer and then eluted with 5 CV elution buffer. The eluted protein was dialysed against 50 mM tris-pH8, 150 mM NaCl, 2 mM TCEP overnight (snakeskin MWCO 3500) and the his-tags were cleaved with thrombin for protein. Samples were then concentrated (Amicon MWCO 10,000) and further purified by gel filtration using Superdex 75 16/60 before being concentrated to 5 mg/ml.

Extraction

Buffers: 50 mM tris-pH8, 300 mM NaCl, 5 mM imidazole, 2 mM TCEP.

Procedure: Cell pastes were resuspended in 20 fold volumes of lysis buffer (50 mM tris-pH8, 300 mM NaCl, 5 mM imidazole, 2 mM TCEP) supplemented with benzonase and protease inhibitors. Cell suspensions were sonicated by 10 mins at 5" on 7" off duty cycle at 120W output power.

Concentration:Concentration used for crystallization : native protein: 3.5 mg/mL

Ligand: Zn, 3-[6-Oxo-3-(3-pyridinyl)-1(6H)-pyridazinyl]propanoic acid

Structure Determination

MassSpec:11920.62 g/mol

Crystallization:The apo crystal structure of the HDAC6 ZnF-UBD 1109-1215 was previously solved at SGC Toronto (PDB ID: 3C5K). These crystals can be obtained by mixing the protein solution at 3.5 mg/ml 1:1 with 3.5M sodium formate, 0.1M bis tris propane, 5% ethylene glycol, pH7 using the vapour diffusion method at room temperature. In this crystal form, the primary binding pocket is occluded by the C-terminus of the adjacent protein molecule in the crystal lattice, although a secondary adjacent site is exposed to the solvent. These crystals can be used to seed for the desired crystal form for fragment screening. Diluting a 1 µl drop containing these crystals 1:10,000 with mother liquor and vortexing the sample vigourously yields a seed mix. HDAC6 ZF UBD 1109-1213 can then be crystallised in a high salt condition containing 2M Na formate, 0.1M Na acetate pH4.6, 5% ethylene glycol, again by the vapour diffusion method. 500nl of protein are added to 400nl mother liquor and then 100nl seed mix per drop, typically plates are set up using a mosquito. These crystals have a solvent exposed ubiquitin binding pocket, amenable to soaking with 5 % DMSO-compound stock for 3 hours and can be cryo cooled in liquid nitrogen without additional cryo-protection.