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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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Ribbon representations of superimposed ATPBDs indicating conformational changesA. fulgidus CopB apo-ATPBD (PDB code 3SKX) (in green) is superimposed with (A) Apo SERCA1 (PDB code 1SU4), (B) A. fulgidus CopA–AMPPCP (PDB code 3A1C), (C) SERCA1-AMPPCP (PDB code 1VFP) and (D) L. pneumophila Lp-CopA, AlF4 (3RFU). The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera.
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Figure 4: Ribbon representations of superimposed ATPBDs indicating conformational changesA. fulgidus CopB apo-ATPBD (PDB code 3SKX) (in green) is superimposed with (A) Apo SERCA1 (PDB code 1SU4), (B) A. fulgidus CopA–AMPPCP (PDB code 3A1C), (C) SERCA1-AMPPCP (PDB code 1VFP) and (D) L. pneumophila Lp-CopA, AlF4 (3RFU). The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera.

Mentions: Previously determined structures of ATPBDs have been observed in both open and closed conformations in which the P- and N-domains are reoriented with respect to each other. However, the physiological factors that influence movement of the domains are still under discussion, and in the observed crystal structures it is possible that crystal packing forces have some influence. The CopB apo-ATPBD determined here was found in the open conformation, similar to the apo-SERCA1 structure (PDB code 1SU4) [13] (Figure 4). The open conformation of the apo-state makes sense with the expectation that this conformation would allow access for nucleotide binding. The two ATPBDs that were co-crystallized with non-hydrolysable nucleotides, A. fulgidus CopA–AMPPCP (PDB code 3A1C) [25] and SERCA1–AMPPCP (PDB code 1VFP) [17], were both observed in the closed conformation, suggesting that the P- and N-domains of the ATPBD move to facilitate simultaneous contacts from both domains with the nucleotide (Figure 4). After phosphorylation of the aspartate in the P-domain, the reopening of the P- and N-domains is necessary for the release of ADP. Thus, the open conformation is poised for subsequent product release after dephosphorylation, as we have observed here in the phosphate-bound structure of the CopB ATPBD. The open conformation is also seen in the Lp-CopA structure with the product analogue AlF4 [29] (Figure 4).


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 representations of superimposed ATPBDs indicating conformational changesA. fulgidus CopB apo-ATPBD (PDB code 3SKX) (in green) is superimposed with (A) Apo SERCA1 (PDB code 1SU4), (B) A. fulgidus CopA–AMPPCP (PDB code 3A1C), (C) SERCA1-AMPPCP (PDB code 1VFP) and (D) L. pneumophila Lp-CopA, AlF4 (3RFU). The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera.
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Related In: Results  -  Collection

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Figure 4: Ribbon representations of superimposed ATPBDs indicating conformational changesA. fulgidus CopB apo-ATPBD (PDB code 3SKX) (in green) is superimposed with (A) Apo SERCA1 (PDB code 1SU4), (B) A. fulgidus CopA–AMPPCP (PDB code 3A1C), (C) SERCA1-AMPPCP (PDB code 1VFP) and (D) L. pneumophila Lp-CopA, AlF4 (3RFU). The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera.
Mentions: Previously determined structures of ATPBDs have been observed in both open and closed conformations in which the P- and N-domains are reoriented with respect to each other. However, the physiological factors that influence movement of the domains are still under discussion, and in the observed crystal structures it is possible that crystal packing forces have some influence. The CopB apo-ATPBD determined here was found in the open conformation, similar to the apo-SERCA1 structure (PDB code 1SU4) [13] (Figure 4). The open conformation of the apo-state makes sense with the expectation that this conformation would allow access for nucleotide binding. The two ATPBDs that were co-crystallized with non-hydrolysable nucleotides, A. fulgidus CopA–AMPPCP (PDB code 3A1C) [25] and SERCA1–AMPPCP (PDB code 1VFP) [17], were both observed in the closed conformation, suggesting that the P- and N-domains of the ATPBD move to facilitate simultaneous contacts from both domains with the nucleotide (Figure 4). After phosphorylation of the aspartate in the P-domain, the reopening of the P- and N-domains is necessary for the release of ADP. Thus, the open conformation is poised for subsequent product release after dephosphorylation, as we have observed here in the phosphate-bound structure of the CopB ATPBD. The open conformation is also seen in the Lp-CopA structure with the product analogue AlF4 [29] (Figure 4).

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