Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.
Bottom Line: Here, we demonstrate the use of NMR spectroscopy to characterise binding of ammonium ions to two different enzymes: human histone deacetylase 8 (HDAC8), which is activated allosterically by potassium, and the bacterial Hsp70 homologue DnaK, for which potassium is an integral part of the active site.Ammonium activates both enzymes in a similar way to potassium, thus supporting this non-invasive approach.Furthermore, we present an approach to map the observed binding site onto the structure of HDAC8.
Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). firstname.lastname@example.org.Show MeSH
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Mentions: The 42 kDa human HDAC8 enzyme is activated by the binding of potassium ions. In addition to the catalytically important Zn2+ ion at the active site, crystal structures of HDAC8 reveal two MVC binding sites (Figure 1 A–C), as has been observed in most structurally characterised HDACs and HDAC-related deacetylases. Biochemical characterisation and computational approaches have highlighted the importance of potassium binding for activity and potentially even for regulation of this enzyme. Specifically, the effect of KCl concentration on HDAC8 activity was investigated by Fierke and co-workers using HDAC8 with two different divalent metal ions (Co2+ and Zn2+) bound at the active site. These experiments revealed a bell-shaped activity profile, with an activating effect at lower concentrations of K+ and an inactivating effect as the concentration was increased. The resulting activation (K1/2,act) and inhibition (K1/2,inhib) constants, which describe the apparent dissociation constants for an activating and an inhibiting binding site, respectively, were K1/2,act=14 mm and K1/2,inhib=130 mm for Zn2+-bound HDAC8.
Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). email@example.com.