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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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Limited proteolysis of the ATPBD of A. fulgidus CopB(A) SDS/PAGE gel of the proteolysis reaction performed in the absence of AMPPCP. (B) Total intensities of the two prominent bands (full-length protein and Fragment A) in the gel plotted as a function of time. (C) SDS/PAGE gel of the reaction performed in the presence of 5 mM AMPPCP. (D) Total intensities of the three prominent bands (full-length protein and Fragments B and C).
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Figure 5: Limited proteolysis of the ATPBD of A. fulgidus CopB(A) SDS/PAGE gel of the proteolysis reaction performed in the absence of AMPPCP. (B) Total intensities of the two prominent bands (full-length protein and Fragment A) in the gel plotted as a function of time. (C) SDS/PAGE gel of the reaction performed in the presence of 5 mM AMPPCP. (D) Total intensities of the three prominent bands (full-length protein and Fragments B and C).

Mentions: To avoid the potential effects of crystal packing forces on the conformation of the ATPBD, we examined the conformation of the CopB ATPBD in solution using limited proteolysis in the absence and presence of nucleotide. With this approach, differences in the number and/or size of fragments produced from proteolysis reflect the conformation of the different states. After proteolysis of the ATPBD in the absence of AMPPCP, two major species were observed (Figure 5). One species is the full-length protein and the other is a smaller fragment that migrates between the 17 and 26 kDa marker (labelled as fragment A in Figure 5). In the presence of 5 mM AMPPCP, two major proteolysis products were observed (labelled as fragments B and C in Figure 5), both of which are different from the major fragment observed in the apo-state. Furthermore, in the presence of nucleotide the full-length ATPBD predominates during the 3 h course of the experiment, indicating that the rate of proteolysis is significantly decreased when a nucleotide is present. There are two major conclusions from this experiment. Firstly, different cleavage patterns indicate different predominant conformations for the apo- and nucleotide-bound states. Secondly, the decrease in cleavage rate indicates overall stability/rigidity conferred on the protein when bound to AMPPCP. This is the first demonstration of conformational changes of the ATPBD in solution, and supports the hypothesis that the P- and N-domains adopt a closed conformation in the presence of nucleotide.


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)

Limited proteolysis of the ATPBD of A. fulgidus CopB(A) SDS/PAGE gel of the proteolysis reaction performed in the absence of AMPPCP. (B) Total intensities of the two prominent bands (full-length protein and Fragment A) in the gel plotted as a function of time. (C) SDS/PAGE gel of the reaction performed in the presence of 5 mM AMPPCP. (D) Total intensities of the three prominent bands (full-length protein and Fragments B and C).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475447&req=5

Figure 5: Limited proteolysis of the ATPBD of A. fulgidus CopB(A) SDS/PAGE gel of the proteolysis reaction performed in the absence of AMPPCP. (B) Total intensities of the two prominent bands (full-length protein and Fragment A) in the gel plotted as a function of time. (C) SDS/PAGE gel of the reaction performed in the presence of 5 mM AMPPCP. (D) Total intensities of the three prominent bands (full-length protein and Fragments B and C).
Mentions: To avoid the potential effects of crystal packing forces on the conformation of the ATPBD, we examined the conformation of the CopB ATPBD in solution using limited proteolysis in the absence and presence of nucleotide. With this approach, differences in the number and/or size of fragments produced from proteolysis reflect the conformation of the different states. After proteolysis of the ATPBD in the absence of AMPPCP, two major species were observed (Figure 5). One species is the full-length protein and the other is a smaller fragment that migrates between the 17 and 26 kDa marker (labelled as fragment A in Figure 5). In the presence of 5 mM AMPPCP, two major proteolysis products were observed (labelled as fragments B and C in Figure 5), both of which are different from the major fragment observed in the apo-state. Furthermore, in the presence of nucleotide the full-length ATPBD predominates during the 3 h course of the experiment, indicating that the rate of proteolysis is significantly decreased when a nucleotide is present. There are two major conclusions from this experiment. Firstly, different cleavage patterns indicate different predominant conformations for the apo- and nucleotide-bound states. Secondly, the decrease in cleavage rate indicates overall stability/rigidity conferred on the protein when bound to AMPPCP. This is the first demonstration of conformational changes of the ATPBD in solution, and supports the hypothesis that the P- and N-domains adopt a closed conformation in the presence of nucleotide.

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