Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations

Abstract

The metal-dependent histone deacetylases (HDACs) catalyze hydrolysis of acetyl groups from acetyllysine side chains and are targets of cancer therapeutics. Two bound monovalent cations (MVCs) of unknown function have been previously observed in crystal structures of HDAC8; site 1 is near the active site, whereas site 2 is located >20 Å from the catalytic metal ion. Here we demonstrate that one bound MVC activates catalytic activity (K 1/2 ‫؍‬ 3.4 mM for K ؉ ), whereas the second, weakerbinding MVC (K 1/2 ‫؍‬ 26 mM for K ؉ ) decreases catalytic activity by 11-fold. The weaker binding MVC also enhances the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid by 5-fold. The site 1 MVC is coordinated by the side chain of Asp-176 that also forms a hydrogen bond with His-142, one of two histidines important for catalytic activity. The D176A and H142A mutants each increase the K 1/2 for potassium inhibition by >40-fold, demonstrating that the inhibitory cation binds to site 1. Furthermore, the MVC inhibition is mediated by His-142, suggesting that this residue is protonated for maximal HDAC8 activity. Therefore, His-142 functions either as an electrostatic catalyst or a general acid. The activating MVC binds in the distal site and causes a time-dependent increase in activity, suggesting that the site 2 MVC stabilizes an active conformation of the enzyme. Sodium binds more weakly to both sites and activates HDAC8 to a lesser extent than potassium. Therefore, it is likely that potassium is the predominant MVC bound to HDAC8 in vivo.

Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

“…2B). The HDAC8 H143A mutant has an almost complete loss of activity in contrast to the residual activity of an H142A mutant, in concordance with the proposed model of action (Gantt et al 2010). Furthermore, quantum mechanical/molecular mechanical molecular dynamics simulations suggest that a neutral H143 first serves as the general base to accept a proton from the zincbound water molecule in the initial rate-determining nucleophilic attack step, and then shuttles it to the amide nitrogen atom to facilitate the cleavage of the amide bond (Wu et al 2011).…”

K + Efflux Is Required for Histone H3 Dephosphorylation by Listeria monocytogenes Listeriolysin O and Other Pore-Forming Toxins

“…Of note, the crystal structures of several histone deacetylases identified a K ϩ ion in their structure (5,31). Furthermore, K ϩ regulates the activity of histone deacetylase 8, rendering this enzyme's activity sensitive to changes in the cellular K ϩ concentration (8). Interestingly, we have shown that LLO-mediated dePH3 correlates with a deacetylation of histone H4, and therefore, the two events could be linked (11).…”

Histone deacetylases: structural determinants of inhibitor selectivity

“…These residues are less conserved than those of site 1 ( Figure S1 in the supplementary material online). The role of these two metal binding sites was investigated in HDAC8 [65] and found to be associated with enzyme activation or inhibition. Kinetic studies demonstrated that HDAC8 is inactive in the absence of added KCl or NaCl and that the enzyme shows higher activity when bound to K + rather than to Na + , the former ion being generally more abundant than the latter in the cellular cytoplasm.…”