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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 the ATPase domain of Hsp70 (PDB ID: 1HPM):[2] purple spheres: K+, green sphere: Mg2+, yellow sticks: ADP and Pi. A Ligplot analysis showing two-dimensional representations of the binding sites is shown in Figure S2. B) 1H,15N HSQC spectra and 1D projections (above) with the ABD of bacterial Hsp70 homologue DnaK. Red spectra: DnaK-ABD (100 μm) in 30 mm Tris⋅HCl, 150 mm15NH4Cl, 5 mm MgCl2 and 1 mm DTT, pH 7.5. Blue spectra: the same sample after addition of 0.5 mm ADP and 50 mm14NH4H2PO4. Note: because of the low pH of the 14NH4H2PO4 stock solution, Tris base was also added to achieve pH 7.5, thus increasing the total Tris concentration to 75 mm.
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fig03: A) Crystal structure of the ATPase domain of Hsp70 (PDB ID: 1HPM):[2] purple spheres: K+, green sphere: Mg2+, yellow sticks: ADP and Pi. A Ligplot analysis showing two-dimensional representations of the binding sites is shown in Figure S2. B) 1H,15N HSQC spectra and 1D projections (above) with the ABD of bacterial Hsp70 homologue DnaK. Red spectra: DnaK-ABD (100 μm) in 30 mm Tris⋅HCl, 150 mm15NH4Cl, 5 mm MgCl2 and 1 mm DTT, pH 7.5. Blue spectra: the same sample after addition of 0.5 mm ADP and 50 mm14NH4H2PO4. Note: because of the low pH of the 14NH4H2PO4 stock solution, Tris base was also added to achieve pH 7.5, thus increasing the total Tris concentration to 75 mm.

Mentions: Another example of a potassium-binding enzyme is the molecular chaperone Hsp70. Two potassium ions in the ATP-binding domain (Figure 3 A) have been identified to be crucial for the ATPase cycle,[6b] which in turn regulates the binding and release of substrate proteins.[13] In a similar way to that for HDAC8, these potassium ions can be replaced by ammonium, thereby resulting in approximately half of the ATPase activity observed with potassium.[6b] Based on our success in characterising the potassium binding sites of HDAC8 with 15NH4+, we tested the general applicability of our approach by probing the potassium binding sites of the 41 kDa ATP-binding domain of DnaK, a bacterial Hsp70 homologue from Thermus thermophilus.


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 the ATPase domain of Hsp70 (PDB ID: 1HPM):[2] purple spheres: K+, green sphere: Mg2+, yellow sticks: ADP and Pi. A Ligplot analysis showing two-dimensional representations of the binding sites is shown in Figure S2. B) 1H,15N HSQC spectra and 1D projections (above) with the ABD of bacterial Hsp70 homologue DnaK. Red spectra: DnaK-ABD (100 μm) in 30 mm Tris⋅HCl, 150 mm15NH4Cl, 5 mm MgCl2 and 1 mm DTT, pH 7.5. Blue spectra: the same sample after addition of 0.5 mm ADP and 50 mm14NH4H2PO4. Note: because of the low pH of the 14NH4H2PO4 stock solution, Tris base was also added to achieve pH 7.5, thus increasing the total Tris concentration to 75 mm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4016748&req=5

fig03: A) Crystal structure of the ATPase domain of Hsp70 (PDB ID: 1HPM):[2] purple spheres: K+, green sphere: Mg2+, yellow sticks: ADP and Pi. A Ligplot analysis showing two-dimensional representations of the binding sites is shown in Figure S2. B) 1H,15N HSQC spectra and 1D projections (above) with the ABD of bacterial Hsp70 homologue DnaK. Red spectra: DnaK-ABD (100 μm) in 30 mm Tris⋅HCl, 150 mm15NH4Cl, 5 mm MgCl2 and 1 mm DTT, pH 7.5. Blue spectra: the same sample after addition of 0.5 mm ADP and 50 mm14NH4H2PO4. Note: because of the low pH of the 14NH4H2PO4 stock solution, Tris base was also added to achieve pH 7.5, thus increasing the total Tris concentration to 75 mm.
Mentions: Another example of a potassium-binding enzyme is the molecular chaperone Hsp70. Two potassium ions in the ATP-binding domain (Figure 3 A) have been identified to be crucial for the ATPase cycle,[6b] which in turn regulates the binding and release of substrate proteins.[13] In a similar way to that for HDAC8, these potassium ions can be replaced by ammonium, thereby resulting in approximately half of the ATPase activity observed with potassium.[6b] Based on our success in characterising the potassium binding sites of HDAC8 with 15NH4+, we tested the general applicability of our approach by probing the potassium binding sites of the 41 kDa ATP-binding domain of DnaK, a bacterial Hsp70 homologue from Thermus thermophilus.

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