Inactivating mutation in histone deacetylase 3 stabilizes its active conformation

Abstract

Histone deacetylases (HDACs), together with histone acetyltransferases (HATs), regulate gene expression by modulating the acetylation level of chromatin. HDAC3 is implicated in many important cellular processes, particularly in cancer cell proliferation and metastasis, making inhibition of HDAC3 a promising epigenetic treatment for certain cancers. HDAC3 is activated upon complex formation with both inositol tetraphosphate (IP4) and the deacetylase-activating domain (DAD) of multi-protein nuclear receptor corepressor complexes. In previous studies, we have shown that binding of DAD and IP4 to HDAC3 significantly restricts its conformational space towards its stable ternary complex conformation, and suggest this to be the active conformation. Here, we report a single mutation of HDAC3 that is capable of mimicking the stabilizing effects of DAD and IP4, without the presence of either. This mutation, however, results in a total loss of deacetylase activity, prompting a closer evaluation of our understanding of the activation of HDAC3.

Keywords: HDAC3; R265P; allostery; conformational selection; molecular recognition.

Figure 1

Cartoon representation of HDAC3 (green ribbon) bound to DAD (purple ribbon) and IP4 (sticks), from Protein Data Bank (PDB) identification code: 4A69. Arrow indicates the location of the active site. For reference, some loops and helices are identified, as well as the location of the R265 residue.

Figure 2

Principal component analysis of HDAC3. (A) apo HDAC3, (B) IP4-bound HDAC3, (C) DAD-bound HDAC3, (D) IP4- and DAD-bound HDAC3, and (E) apo HDAC3^R265P mutant dynamics are each projected onto PC1:PC2 space of the apo HDAC3^WT. A white cross in each panel indicates the projection of the HDAC3:IP4:DAD crystal structure (PDB ID: 4A6910).

Figure 3

Analysis of mutant and complexed HDAC3. Differences between per-residue RMSF values (Å) are shown for (from top to bottom) HDAC3:IP4:DAD, HDAC3:DAD, HDAC3:IP4, and HDAC3^R265P, with reference to the apo HDAC3^WT RMSF values. Negative values correspond to stabilized regions.

Figure 4

Geometry of active site channel. (A) Two-dimensional histograms showing the distribution of radii along the channel length for the apo, IP4-bound, DAD-bound, and ternary WT simulations, as well as for the apo R265P mutant. (B) The stable channel leading to the active site, from simulations of the HDAC3:IP4:DAD complex is shown schematically, with average diameters indicated along the channel length.

Figure 5

Population analysis of Tyr298 dihedral angle. (A) Normalized probability of the side chain dihedral angle shows two states (inward and outward) sampled in the HDAC3 simulations. (B) Cartoon representation of HDAC3 from the HDAC3:IP4:DAD (black) and apo (green) simulations are superimposed to highlight the two Tyr298 conformations. An arbitrary acetyllysine peptide substrate was docked into the active site to show the functional relevance of the inward Tyr298 conformation (hydrogen atoms are not shown).