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 A 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, weaker-binding 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 > or = 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.

FIGURE 1.

HDAC8 active site and monovalent site 1. The site 1 MVC is coordinated by the backbone carbonyl oxygens of Asp-176, Asp-178, His-180, and Leu-200 and by the side chains of Asp-176 and Ser-199. The carboxylate of Asp-176 also forms a hydrogen bond with His-142. With MVC 1 bound, as shown, the catalytic activity is less than maximal, likely due to the deprotonation of His-142.

FIGURE 2.

HDAC8 activation and inhibition by monovalent ions. Co(II)-wt-HDAC8 was incubated with KCl (●) or NaCl (□) for 1 h on ice before assaying activity with 0.4 μm enzyme and 50 μm Fluor de Lys HDAC8 substrate in 25 mm Tris pH 8.0, 25 °C. Initial velocities were determined based on changes in fluorescence, and Equations 1 and 2 are fit to the resulting rates at varying KCl and NaCl, respectively, yielding the K_1/2,act and K_1/2,inhib values shown in Table 1.

SCHEME 1.

MVC binding of HDAC8. The first MVC activates HDAC8, whereas the second MVC partially inhibits activity (k_obs1 > k_obs2).

FIGURE 3.

Dependence of the activity of HDAC8 mutants on KCl. Initial rates for HDAC8-catalyzed deacetylation of Fluor de Lys H4-Ack16 (wt, H142A, D176N) or HDAC8 (D176A) substrate were measured at 25 °C in 25 mm Tris pH 8.0 with 0.03–2000 mm KCl for the Co(II)-HDAC8 mutants H142A (●, 10 μm enzyme, 50 μm substrate), D176N (□, 10 μm enzyme, 200 μm substrate), and D176A (△, 20 μm enzyme, 150 μm substrate). Equation 2 is fit to the data for for D176N and D176A, and Equation 3 is fit to the data for H142A with the resulting apparent binding affinities listed in Table 1.

FIGURE 4.

Time dependence of HDAC8 activation by KCl. Co(II)-wt-HDAC8 (0.44 μm) was incubated with 5 mm KCl in 25 mm Tris, pH 8.0, at 25 °C for the indicated length of time before assaying activity with 0.4 μm HDAC8 and 50 μm Fluor de Lys HDAC8 substrate. A single exponential curve (Equation 5) was fit to these data to determine the observed rate constant, k_obs, for activation of Co(II)-HDAC8 by KCl.

FIGURE 5.

Dependence of the SAHA inhibition constant for HDAC8 on KCl. Co(II)-HDAC8 (0.2 μm) was incubated with SAHA at a given K⁺ concentration. Initial rates for deacetylation of Fluor de Lys HDAC8 (50 μm) were measured at 25 °C in 25 mm Tris, pH 8.0, containing 0.2–5 μm SAHA and 0.04–200 mm KCl. The SAHA inhibition constant at each K⁺ concentration was obtained from a fit of Equation 6 to the dependence of activity on the concentration of SAHA. Equations 7 and 8 are fit to these data to determine affinity of SAHA for E, EK, and EK₂.

SCHEME 2.

Coupled binding of SAHA and K⁺ to HDAC8. The influence of potassium binding to MVC1 and MVC2 sites of wt-HDAC8 on the affinity of this enzyme for SAHA is described by this simplified three-state scheme where SAHA binds to the three major enzyme species, HDAC8 with no bound K⁺ (E), K⁺ bound to MVC site 2 (EK), and K⁺ bound to both MVC sites 1 and 2 (EK₂). For simplicity, this scheme does not include SAHA binding to HDAC8 with K⁺ bound to MVC2, which constitutes only a minor fraction of the total enzyme. Fits to this model demonstrate that the affinity of SAHA decreases as K⁺ dissociates from the enzyme.

SCHEME 3.

A conformational change may explain the slow activation of HDAC8 by KCl. In this model E and E* are two slowly interconverting conformations of HDAC8. Until M⁺ binds, the enzyme is inactive, and E* must convert to E before it can bind M⁺. Rapid binding of M⁺ to E activates catalysis, as shown in Scheme 1.

FIGURE 6.

Inhibitory K⁺ binding influences His-142 protonation. The protonated form of His-142 is expected to be thermodynamically more favored in the absence of the inhibitory K⁺ (upper panel) than when K⁺ is bound (lower panel).