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Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

ABSTRACT: Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The structures presented, coupled with activity data, provide the molecular basis for the intrinsically low enzymatic activity of class IIa HDACs toward acetylated lysines and reveal active site features that may guide the design of class-specific inhibitors. In addition, these structures reveal a conformationally flexible structural zinc-binding domain conserved in all class IIa enzymes. Importantly, either the mutation of residues coordinating the structural zinc ion or the binding of a class IIa selective inhibitor prevented the association of HDAC4 with the N-CoR.HDAC3 repressor complex. Together, these data suggest a key role of the structural zinc-binding domain in the regulation of class IIa HDAC functions.

SUBMITTER: Bottomley MJ

PROVIDER: S-EPMC3258910 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

Bottomley Matthew J MJ Lo Surdo Paola P Di Giovine Paolo P Cirillo Agostino A Scarpelli Rita R Ferrigno Federica F Jones Philip P Neddermann Petra P De Francesco Raffaele R Steinkühler Christian C Gallinari Paola P Carfí Andrea A

The Journal of biological chemistry 20080708 39

Histone deacetylases (HDACs) regulate chromatin status and gene expression, and their inhibition is of significant therapeutic interest. To date, no biological substrate for class IIa HDACs has been identified, and only low activity on acetylated lysines has been demonstrated. Here, we describe inhibitor-bound and inhibitor-free structures of the histone deacetylase-4 catalytic domain (HDAC4cd) and of an HDAC4cd active site mutant with enhanced enzymatic activity toward acetylated lysines. The s ...[more]

PMID: 18614528

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Project description:BACKGROUND: The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded ? sheet and three long ? helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?" METHODS: We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded ? sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3 day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. RESULTS: This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional "domain" found to date for the matrixin family. CONCLUSIONS: The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.

Dataset Information

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

Publications

Structural and functional analysis of the human HDAC4 catalytic domain reveals a regulatory structural zinc-binding domain.

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