Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.

Nicolas D Werbeck,Jochen Reinstein,D Flemming Hansen,John Kirkpatrick

doi:10.1002/cbic.201300700

Abstract

A variety of enzymes are activated by the binding of potassium ions. The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. In these cases, ammonium can be used as a proxy for potassium to characterise potassium binding sites in enzymes: the 1H,15N spin-pair of enzyme-bound 15NH4+ can be probed by 15N-edited heteronuclear NMR experiments. 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. Our method for mapping the binding site is general and does not require chemical shift assignment of the enzyme resonances.

Highlights

Many enzymes are activated by specific binding of one or more monovalent cations (MVCs); these either contribute directly to the catalysis at the active site or indirectly modulate the activity by binding to an allosteric site
The 42 kDa human histone deacetylase 8 (HDAC8) enzyme is activated by the binding of potassium ions.[5]
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.[5]

Summary

CHEMBIOCHEM FULL PAPERS

The potassium binding sites of these enzymes are very specific, but ammonium ions can often replace potassium ions in vitro because of their similar ionic radii. 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 Hsp homologue DnaK, for which potassium is an integral part of the active site. Ammonium activates both enzymes in a similar way to potassium, supporting this non-invasive approach. Our method for mapping the binding site is general and does not require chemical shift assignment of the enzyme resonances

Introduction

Results and Discussion

Binding of ammonium to DnaK

Mapping the MVC binding sites

Conclusions

Experimental Section