Human histone deacetylase 6 zinc finger domain

Target ID:

HDAC6

Aliases:

FLJ16239HD6JM21

Deposition Date:

Thursday 31st January 2008

Families:

Ubiquitin Related Biology

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

3C5K

Authors:

Dong A, Ravichandran M, Schuetz A, Loppnau P, Li Y, MacKenzie F, Kozieradzki I, Edwards AM, Arrowsmith CH, Weigelt J, Bountra C, Bochkarev A, Dhe-Paganon S, Min J, Ouyang H

Site:

Toronto

ICM Datapack:

ICM Datapack

3C5K

tabs

Structure Details

Acetylation of histones, as well as non-histone proteins, plays important roles in regulating various cellular processes. Dynamic control of protein acetylation levels in vivo occurs through the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) (Sadoul et al 2008). HDACs have been grouped in distinct families according to the similarity of their sequence to a yeast founding member. Class I members, such as HDAC1, HDAC2, HDAC3, and HDAC8, are well-known enzymatic transcriptional corepressors homologous to yeast Rpd3. Class II members, including HDAC4, HDAC5, HDAC6, HDAC7, HDAC9,and HDAC10, possess domains similar to the deacetylase domain of yeast Hdal. Class III members, such as SIRT proteins, are related to yeast SIR2 deacetylase, which is an NAD+ dependant enzyme (Mariadason 2008, Sadoul et al 2008).

HDAC6 is a unique member of HDAC family with specific structural and functional features. It is actively or stably maintained in the cytoplasm and is the only member that harbors a full duplication of its deacetylase homology region followed by a specific ubiquitoin-binding domain at the C-terminus end. HDAC6 also has a dynein motor binding region beside the deacetylase catalytic domain. Accordingly, this deacetylase functions at the heart of a cellular regulatory mechanism capable of recognizing and transporting polyubiquitinized unfolded protein to aggresome through the microtubule network (Kawaguchi et al 2003). Moreover, HDAC6 has also found to interact with deubiquitinases and regulating HSP90 chaperone activity. Therefore, this multifunctional protein plays a critical role in mediating and coordinating various cellular events in response to different stressful stimuli (Bali et al 2005, Boyault et al 2007).

We here present the crystal structure of zinc finger domain of human HDAC6. This domain mainly consists of five antiparallel β strands, which are sandwiched by two long α-helices. There is also a short α-helix on the same side of β-sheet as the second α-helix. This compact domain features three well defined zinc-binding sites formed by eight cysteine and four histidine residues. Each zinc-binding site contains four residues with their respective side chains positioned ideally for tetrahedral zinc coordination. These zinc ions have been found to be crucial for the correct folding of the protein. The overall fold of this domain resembles the structures of ubiquitin-binding domains of IsoT and Ubp-M, which further provides insight into how this domain binds ubiquitn (Reyes-Turcu et al 2006, Pai et al 2007).

Materials and Methods

HDAC6

PDB:3C5K
Entry Clone Accession:NP_006035
Entry Clone Source:MGC AT36-C10 (BC013737)
SGC Clone Accession:HDAC6_42; plate APC051F10
Tag:N-terminal tag: MGSSHHHHHHSSGLVPRGS
Host:BL21 (DE3)
Vector:pET28a-LIC

Sequence: MGSSHHHHHHSSGLVPRGSPLPWCPHLVAVCPIPAAGLDVTQPCGDCGTIQENWVCLSCYQVYCGRYINGHMLQHHGNSGHPLVLSYIDLSAWCYYCQAYVHHQALLDVKNIAHQNKFGEDMPHPH

Growth

Medium:TB
Procedure: A 250 ml flask containing LB (Sigma L7658) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 degC.

Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 ml overnight LB culture, and incubated at 37 degC. The temperature of the media was reduced to 15 degC one hour prior to induction and induced at OD(600) = 6 with 100 uM isopropyl-thio-ß-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC, and cell pellets collected by centrifugation and frozen at -80 degC.

Purification

Procedure:

IMAC: Unclarified lysate was mixed with 2-3 mL of Ni-NTA superflow Resin (Qiagen) per 40 mL lysate. The mixture was incubated with mixing for at least 45 minutes at 4 degC. The mixture was than loaded onto an empty comlum (BioRad) and washed with 100 ml wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280. Thrombin was added to eluted protein at 1 unit per mg of eluted protein and dialyze against gel filtration buffer overnight to remove His-tag.

Gel filtration chromatography: An XK 26x65 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 2.5mL/min. The dialyzed sample from the IMAC step (approx. 15 mL) was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

ProcedureFrozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 15 mg/mL.

Structure Determination

Crystallization:Crystals of the HDAC6 zinc finger domain were grown at 298 K using the hanging drop method by mixing equal volumes of 3.5M Na Formate, 100 mM Bis-Tris pH7.0, and 15 mg/mL protein. The crystals were cryoprotected by dragging through paratone oil.

References

Bali P, Pranpat M, Bradner J, Balasis M, Fiskus W, Guo F, Rocha K, Kumaraswamy S, Boyapalle S, Atadja P, Seto E, Bhalla K. (2005) Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. Biol Chem. 280:26729-34.
Boyault C, Sadoul K, Pabion M, Khochbin S. (2007) HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination. Oncogene. 26:5468-76.
Kawaguchi, Y., Kovacs, J. J., McLaurin, A., Vance, J. M., Ito, A. & Yao, T.-P. (2003). The deacetylase HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress. Cell, 115, 727-738.
Mariadason JM. (2008). HDACs and HDAC inhibitors in colon cancer. Epigenetics. 3(1) [Epub ahead of print] Related Articles, LinksPai MT, Tzeng SR, Kovacs JJ, Keaton MA, Li SS, Yao TP, Zhou P. (2007) Solution structure of the Ubp-M BUZ domain, a highly specific protein module that recognizes the C-terminal tail of free ubiquitin.J Mol Biol. 370:290-302.
Reyes-Turcu FE, Horton JR, Mullally JE, Heroux A, Cheng X, Wilkinson KD. (2006) The ubiquitin binding domain ZnF UBP recognizes the C-terminal diglycine motif of unanchored ubiquitin. Cell. 124:1197-208.
Sadoul K, Boyault C, Pabion M, Khochbin S. (2008) Regulation of protein turnover by acetyltransferases and deacetylases. Biochimie. 90:306-12.