title: Using ¹⁵N-ammonium to characterise and map potassium binding sites in proteins by NMR spectroscopy.
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 (1) H,(15) N spin-pair of enzyme-bound (15) NH4 (+) can be probed by (15) N-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.
authors: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). d.hansen@ucl.ac.uk.
authors: Nicolas D
authors: ND
authors: Werbeck
authors: John
authors: J
authors: Kirkpatrick
authors: Jochen
authors: J
authors: Reinstein
authors: D Flemming
authors: DF
authors: Hansen
mesh_terms: D064751:Ammonium Compounds / Q000737:chemistry*; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006655:Histone Deacetylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D056004:Molecular Dynamics Simulation; D009587:Nitrogen Isotopes / Q000737:chemistry; D011188:Potassium / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D064751:Ammonium Compounds; D018840:HSP70 Heat-Shock Proteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012097:Repressor Proteins; C409470:HDAC8 protein, human; D006655:Histone Deacetylases; D011188:Potassium
keywords: HDAC; Hsp70; NMR spectroscopy; ammonium; enzyme catalysis; potassium binding