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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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Conformational changes in CopB ATPBD in solution probed by intrinsic tryptophan fluorescence(A) The single tryptophan in the CopB ATPBD, shown in blue space-filling representation, is located proximal to the hinge region. (B) Decrease in fluorescence emission at 340 nm accompanies the addition of ATP.
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Figure 6: Conformational changes in CopB ATPBD in solution probed by intrinsic tryptophan fluorescence(A) The single tryptophan in the CopB ATPBD, shown in blue space-filling representation, is located proximal to the hinge region. (B) Decrease in fluorescence emission at 340 nm accompanies the addition of ATP.

Mentions: Ligand-induced conformational changes in solution were also examined by intrinsic Trp546 fluorescence experiments. The ATPBD of CopB has one Trp546 residue situated in helix α7, which is proximal to the hinge region (Figure 6A). This residue serves as a natural probe for interdomain conformational changes that result from nucleotide binding. The CopB ATPBD exhibits high affinity for ATP (Kd=34±2 μM) as calculated from the change in fluorescence signal upon the addition of ATP (Supplementary Figure S6 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm). More importantly, addition of ATP to the apo-protein results in a decrease in fluorescence emission (Figure 6B), as would be expected if nucleotide binding results in an increase in solvent accessibility of the Trp546 residue in the nucleotide-bound state compared with the apo state. Modelling of the CopB ATPBD in closed conformation shows that Trp546 would have a significant increase in accessibility in closed conformation as compared with open conformation (Supplementary Figure S7 and Supplementary Table S1 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm). These observations provide further support that the closed conformation is adopted in solution upon nucleotide binding.


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)

Conformational changes in CopB ATPBD in solution probed by intrinsic tryptophan fluorescence(A) The single tryptophan in the CopB ATPBD, shown in blue space-filling representation, is located proximal to the hinge region. (B) Decrease in fluorescence emission at 340 nm accompanies the addition of ATP.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Conformational changes in CopB ATPBD in solution probed by intrinsic tryptophan fluorescence(A) The single tryptophan in the CopB ATPBD, shown in blue space-filling representation, is located proximal to the hinge region. (B) Decrease in fluorescence emission at 340 nm accompanies the addition of ATP.
Mentions: Ligand-induced conformational changes in solution were also examined by intrinsic Trp546 fluorescence experiments. The ATPBD of CopB has one Trp546 residue situated in helix α7, which is proximal to the hinge region (Figure 6A). This residue serves as a natural probe for interdomain conformational changes that result from nucleotide binding. The CopB ATPBD exhibits high affinity for ATP (Kd=34±2 μM) as calculated from the change in fluorescence signal upon the addition of ATP (Supplementary Figure S6 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm). More importantly, addition of ATP to the apo-protein results in a decrease in fluorescence emission (Figure 6B), as would be expected if nucleotide binding results in an increase in solvent accessibility of the Trp546 residue in the nucleotide-bound state compared with the apo state. Modelling of the CopB ATPBD in closed conformation shows that Trp546 would have a significant increase in accessibility in closed conformation as compared with open conformation (Supplementary Figure S7 and Supplementary Table S1 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm). These observations provide further support that the closed conformation is adopted in solution upon nucleotide binding.

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