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Conformations of the apo-, substrate-bound and phosphate-bound ATP-binding domain of the Cu(II) ATPase CopB illustrate coupling of domain movement to the catalytic cycle.

Jayakanthan S, Roberts SA, Weichsel A, Argüello JM, McEvoy MM - Biosci. Rep. (2012)

Bottom Line: The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion.The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation.These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, U.S.A.

ABSTRACT
Heavy metal P1B-type ATPases play a critical role in cell survival by maintaining appropriate intracellular metal concentrations. Archaeoglobus fulgidus CopB is a member of this family that transports Cu(II) from the cytoplasm to the exterior of the cell using ATP as energy source. CopB has a 264 amino acid ATPBD (ATP-binding domain) that is essential for ATP binding and hydrolysis as well as ultimately transducing the energy to the transmembrane metal-binding site for metal occlusion and export. The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion. Through crystal structures of the apo- and phosphate-bound ATPBDs, with limited proteolysis and fluorescence studies of the apo- and substrate-bound states, we show that the isolated ATPBD of CopB cycles from an open conformation in the apo-state to a closed conformation in the substrate-bound state, then returns to an open conformation suitable for product release. The present work is the first structural report of an ATPBD with its physiologically relevant product (phosphate) bound. The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation. These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.

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Related in: MedlinePlus

Ribbon representation of the 2.1 Å structure of A. fulgidus CopB ATPBD with bound phosphateThe insert shows the phosphate anion being stabilized by the conserved HAD motif residues Lys565, Thr391, Thr537, Asp389 and two water molecules. The phosphate anion in the upper panel is displayed as orange and red spheres. The conserved residues are displayed as sticks. The surface diagram in the lower panel illustrates the surface charges on the anion binding pocket, with blue and red surfaces indicating positively and negatively charged surfaces. The anion is displayed as orange and red sticks, whereas the water molecules are displayed as red spheres. The Models were generated using Pymol and UCSF Chimera, respectively.
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Figure 3: Ribbon representation of the 2.1 Å structure of A. fulgidus CopB ATPBD with bound phosphateThe insert shows the phosphate anion being stabilized by the conserved HAD motif residues Lys565, Thr391, Thr537, Asp389 and two water molecules. The phosphate anion in the upper panel is displayed as orange and red spheres. The conserved residues are displayed as sticks. The surface diagram in the lower panel illustrates the surface charges on the anion binding pocket, with blue and red surfaces indicating positively and negatively charged surfaces. The anion is displayed as orange and red sticks, whereas the water molecules are displayed as red spheres. The Models were generated using Pymol and UCSF Chimera, respectively.

Mentions: The ATPBD of A. fulgidus CopB was co-crystallized with a nucleotide (p[NH]ppA). After accounting for the protein, the electron density showed a significant positive peak near the expected nucleotide-binding site. Although this density was too small to accommodate the full nucleotide, a phosphate anion, one of the hydrolysis products of p[NH]ppA, fits very well into this feature with respect to size and chemical interactions with the protein (Figure 3, and Supplementary Figure S4 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm). No other possible anions, such as sulfate, were present in the crystallization conditions or the protein purification buffers, supporting the identification of this feature as a phosphate anion.


Conformations of the apo-, substrate-bound and phosphate-bound ATP-binding domain of the Cu(II) ATPase CopB illustrate coupling of domain movement to the catalytic cycle.

Jayakanthan S, Roberts SA, Weichsel A, Argüello JM, McEvoy MM - Biosci. Rep. (2012)

Ribbon representation of the 2.1 Å structure of A. fulgidus CopB ATPBD with bound phosphateThe insert shows the phosphate anion being stabilized by the conserved HAD motif residues Lys565, Thr391, Thr537, Asp389 and two water molecules. The phosphate anion in the upper panel is displayed as orange and red spheres. The conserved residues are displayed as sticks. The surface diagram in the lower panel illustrates the surface charges on the anion binding pocket, with blue and red surfaces indicating positively and negatively charged surfaces. The anion is displayed as orange and red sticks, whereas the water molecules are displayed as red spheres. The Models were generated using Pymol and UCSF Chimera, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475447&req=5

Figure 3: Ribbon representation of the 2.1 Å structure of A. fulgidus CopB ATPBD with bound phosphateThe insert shows the phosphate anion being stabilized by the conserved HAD motif residues Lys565, Thr391, Thr537, Asp389 and two water molecules. The phosphate anion in the upper panel is displayed as orange and red spheres. The conserved residues are displayed as sticks. The surface diagram in the lower panel illustrates the surface charges on the anion binding pocket, with blue and red surfaces indicating positively and negatively charged surfaces. The anion is displayed as orange and red sticks, whereas the water molecules are displayed as red spheres. The Models were generated using Pymol and UCSF Chimera, respectively.
Mentions: The ATPBD of A. fulgidus CopB was co-crystallized with a nucleotide (p[NH]ppA). After accounting for the protein, the electron density showed a significant positive peak near the expected nucleotide-binding site. Although this density was too small to accommodate the full nucleotide, a phosphate anion, one of the hydrolysis products of p[NH]ppA, fits very well into this feature with respect to size and chemical interactions with the protein (Figure 3, and Supplementary Figure S4 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm). No other possible anions, such as sulfate, were present in the crystallization conditions or the protein purification buffers, supporting the identification of this feature as a phosphate anion.

Bottom Line: The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion.The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation.These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, U.S.A.

ABSTRACT
Heavy metal P1B-type ATPases play a critical role in cell survival by maintaining appropriate intracellular metal concentrations. Archaeoglobus fulgidus CopB is a member of this family that transports Cu(II) from the cytoplasm to the exterior of the cell using ATP as energy source. CopB has a 264 amino acid ATPBD (ATP-binding domain) that is essential for ATP binding and hydrolysis as well as ultimately transducing the energy to the transmembrane metal-binding site for metal occlusion and export. The relevant conformations of this domain during the different steps of the catalytic cycle are still under discussion. Through crystal structures of the apo- and phosphate-bound ATPBDs, with limited proteolysis and fluorescence studies of the apo- and substrate-bound states, we show that the isolated ATPBD of CopB cycles from an open conformation in the apo-state to a closed conformation in the substrate-bound state, then returns to an open conformation suitable for product release. The present work is the first structural report of an ATPBD with its physiologically relevant product (phosphate) bound. The solution studies we have performed help resolve questions on the potential influence of crystal packing on domain conformation. These results explain how phosphate is co-ordinated in ATPase transporters and give an insight into the physiologically relevant conformation of the ATPBD at different steps of the catalytic cycle.

Show MeSH
Related in: MedlinePlus