Crystallographic and biochemical characterization of human histone deacetylase 4 N-terminal glutamine-rich domain.

摘要

The transcription factor Myocyte Enhaner Factor-2 (MEF2) and its coregulators have been shown to regulate gene expression in response to calcium signals and influence a broad range of biological activities in the muscle, immune and nervous systems. Class IIa Histone deacetylases (HDACs), including HDAC4, 5, 7 and 9 have been identified as important factors in repression of MEF2-dependent genes. Intriguingly, this repression function seems to be independent of the HDAC catalytic domain. Sequence alignment analysis reveals that Class IIa HDACs contain an additional conserved N-terminal glutamine-rich extension that confers responsiveness to calcium signals and mediates interactions with transcription factors and co-factors. Glutamine-rich sequences have been found in a variety of eukaryotic proteins, including transcription activators and repressors. Unfortunately, there is no high-resolution structural information available mostly due to their low solubility. In this thesis, the first high-resolution structure of a glutamine-rich domain from human HDAC4 was determined by X-ray crystallography at 2.6 A. The glutamine-rich domain of HDAC4, folds into a straight alpha helix that assembles as a tetramer. In contrast to most coiled coil proteins, the HDAC4 tetramer lacks regularly arranged apolar residues and an extended hydrophobic core. Instead, the protein interfaces consist of multiple hydrophobic patches interspersed with polar interaction networks wherein clusters of glutamines engage in extensive intra- and inter-helical interactions. Moreover, the HDAC4 tetramer undergoes rapid equilibrium with monomer and intermediate species in solution. Structure-guided mutations that expand or disrupt hydrophobic patches drive the equilibrium toward the tetramer or monomer, respectively. Luciferase assays were performed to further confirm that the oligomerization of HDAC4 is relevant to MEF2-dependent gene regulation through the assembly of large nucleoprotein complexes. Thus, a general role of glutamine-rich domains of Class IIa HDACs may be to mediate protein-protein interactions characteristic of a large component of polar interaction networks that may facilitate reversible assembly and disassembly of protein complexes.