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

Werbeck ND, Kirkpatrick J, Reinstein J, Hansen DF - Chembiochem (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). d.hansen@ucl.ac.uk.

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A) Crystal structure of HDAC8 (PDB ID: 2V5W) with the active-site Zn2+ (black sphere) and the two K+ ions (purple spheres). B) Residues within approximately 10 Å of the two potassium binding sites in HDAC8. Histidine residues are shown in cyan. C) Two-dimensional representations of the two binding sites (generated with Ligplot).[12] K+-coordinating residues and atoms are labelled. D) Activity profile as a function of monovalent cation concentration. Concentrations of K+ (red), NH4+ (green) and choline+ (blue) were in 25 mm Tris⋅HCl at pH 8.0. The two-site binding model described previously[5] was used to fit the activity profile for KCl and NH4Cl according to the equation v=v1/(1+K1/2,act/[MVC]+[MVC]/K1/2,inh), where K1/2,act and K1/2,inh are the apparent binding affinities (dissociation constants) for activation and inhibition, v is the relative kcat/KM value, v1 is the kcat/KM value of the fully activated state and [MVC] is the concentration of the respective monovalent cation. kcat/KM of the fully inhibited state was set as zero; data are normalised such that v1 (K+)=1.0. K1/2,act (K+): 45±26 mm, K1/2,inh (K+): 610±530 mm, K1/2,act (NH4+): 25±8 mm, K1/2,inh (NH4+): 2600±1700 mm. Although the errors for the affinities of the inhibitory site are large, these data agree with a bell-shaped activity profile for K+ and NH4+.
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fig01: A) Crystal structure of HDAC8 (PDB ID: 2V5W) with the active-site Zn2+ (black sphere) and the two K+ ions (purple spheres). B) Residues within approximately 10 Å of the two potassium binding sites in HDAC8. Histidine residues are shown in cyan. C) Two-dimensional representations of the two binding sites (generated with Ligplot).[12] K+-coordinating residues and atoms are labelled. D) Activity profile as a function of monovalent cation concentration. Concentrations of K+ (red), NH4+ (green) and choline+ (blue) were in 25 mm Tris⋅HCl at pH 8.0. The two-site binding model described previously[5] was used to fit the activity profile for KCl and NH4Cl according to the equation v=v1/(1+K1/2,act/[MVC]+[MVC]/K1/2,inh), where K1/2,act and K1/2,inh are the apparent binding affinities (dissociation constants) for activation and inhibition, v is the relative kcat/KM value, v1 is the kcat/KM value of the fully activated state and [MVC] is the concentration of the respective monovalent cation. kcat/KM of the fully inhibited state was set as zero; data are normalised such that v1 (K+)=1.0. K1/2,act (K+): 45±26 mm, K1/2,inh (K+): 610±530 mm, K1/2,act (NH4+): 25±8 mm, K1/2,inh (NH4+): 2600±1700 mm. Although the errors for the affinities of the inhibitory site are large, these data agree with a bell-shaped activity profile for K+ and NH4+.

Mentions: The 42 kDa human HDAC8 enzyme is activated by the binding of potassium ions.[5] 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.[10] Biochemical characterisation[5] and computational approaches[11] 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.[5] 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.


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

Werbeck ND, Kirkpatrick J, Reinstein J, Hansen DF - Chembiochem (2014)

A) Crystal structure of HDAC8 (PDB ID: 2V5W) with the active-site Zn2+ (black sphere) and the two K+ ions (purple spheres). B) Residues within approximately 10 Å of the two potassium binding sites in HDAC8. Histidine residues are shown in cyan. C) Two-dimensional representations of the two binding sites (generated with Ligplot).[12] K+-coordinating residues and atoms are labelled. D) Activity profile as a function of monovalent cation concentration. Concentrations of K+ (red), NH4+ (green) and choline+ (blue) were in 25 mm Tris⋅HCl at pH 8.0. The two-site binding model described previously[5] was used to fit the activity profile for KCl and NH4Cl according to the equation v=v1/(1+K1/2,act/[MVC]+[MVC]/K1/2,inh), where K1/2,act and K1/2,inh are the apparent binding affinities (dissociation constants) for activation and inhibition, v is the relative kcat/KM value, v1 is the kcat/KM value of the fully activated state and [MVC] is the concentration of the respective monovalent cation. kcat/KM of the fully inhibited state was set as zero; data are normalised such that v1 (K+)=1.0. K1/2,act (K+): 45±26 mm, K1/2,inh (K+): 610±530 mm, K1/2,act (NH4+): 25±8 mm, K1/2,inh (NH4+): 2600±1700 mm. Although the errors for the affinities of the inhibitory site are large, these data agree with a bell-shaped activity profile for K+ and NH4+.
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Related In: Results  -  Collection

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fig01: A) Crystal structure of HDAC8 (PDB ID: 2V5W) with the active-site Zn2+ (black sphere) and the two K+ ions (purple spheres). B) Residues within approximately 10 Å of the two potassium binding sites in HDAC8. Histidine residues are shown in cyan. C) Two-dimensional representations of the two binding sites (generated with Ligplot).[12] K+-coordinating residues and atoms are labelled. D) Activity profile as a function of monovalent cation concentration. Concentrations of K+ (red), NH4+ (green) and choline+ (blue) were in 25 mm Tris⋅HCl at pH 8.0. The two-site binding model described previously[5] was used to fit the activity profile for KCl and NH4Cl according to the equation v=v1/(1+K1/2,act/[MVC]+[MVC]/K1/2,inh), where K1/2,act and K1/2,inh are the apparent binding affinities (dissociation constants) for activation and inhibition, v is the relative kcat/KM value, v1 is the kcat/KM value of the fully activated state and [MVC] is the concentration of the respective monovalent cation. kcat/KM of the fully inhibited state was set as zero; data are normalised such that v1 (K+)=1.0. K1/2,act (K+): 45±26 mm, K1/2,inh (K+): 610±530 mm, K1/2,act (NH4+): 25±8 mm, K1/2,inh (NH4+): 2600±1700 mm. Although the errors for the affinities of the inhibitory site are large, these data agree with a bell-shaped activity profile for K+ and NH4+.
Mentions: The 42 kDa human HDAC8 enzyme is activated by the binding of potassium ions.[5] 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.[10] Biochemical characterisation[5] and computational approaches[11] 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.[5] 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.

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Structural and Molecular Biology, Division of Biosciences, University College London, Gower Street, London, WC1E 6BT (UK). d.hansen@ucl.ac.uk.

Show MeSH
Related in: MedlinePlus