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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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Secondary structure differences between the P-domains of CopB and Lp-CopA(A) Multiple sequence alignment of the residues corresponding to the loop/helical segment in the P-domains whose structures are known. The secondary structural elements are displayed for CopB (top) and Lp-CopA (bottom). Sequence alignment was performed using CLUSTALW [49]. The solvent accessible residues for CopB (top) and Lp-CopA (bottom) are indicated by bars running parallel to the alignment with colours ranging from white to dark blue. The accessibility information is extracted from the corresponding DSSP file. Blue indicates accessible, cyan indicates intermediate, and white indicates that the residues are buried. This Figure was prepared using the Program ESPript [50]. (B). Cartoon models of CopB (green) and Lp-CopA (blue) superimposed with the insert highlighting the different secondary structure elements between the two structures. The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera [48].
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Figure 2: Secondary structure differences between the P-domains of CopB and Lp-CopA(A) Multiple sequence alignment of the residues corresponding to the loop/helical segment in the P-domains whose structures are known. The secondary structural elements are displayed for CopB (top) and Lp-CopA (bottom). Sequence alignment was performed using CLUSTALW [49]. The solvent accessible residues for CopB (top) and Lp-CopA (bottom) are indicated by bars running parallel to the alignment with colours ranging from white to dark blue. The accessibility information is extracted from the corresponding DSSP file. Blue indicates accessible, cyan indicates intermediate, and white indicates that the residues are buried. This Figure was prepared using the Program ESPript [50]. (B). Cartoon models of CopB (green) and Lp-CopA (blue) superimposed with the insert highlighting the different secondary structure elements between the two structures. The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera [48].

Mentions: In P-type ATPases, the P-domain interacts with the A-domain at different points in the catalytic cycle, as seen in the SERCA1 ATPase structures [17,42,43] and Lp-CopA structures [29]. The surface of the P-domain makes these interactions correspond to the region between β11 and β12 in the CopB structure. In SERCA1 and Lp-CopA (Figure 2), where interdomain contacts are seen, this region is helical. In the CopB ATPBD (Figure 2), as well as the ATPBDs of A. fulgidus CopA [28] and S. solfataricus CopB [24], this region has an irregular loop conformation. It is possible that domain–domain interactions influence the structure in this region of the P-domain, and a helical structure potentially could be adopted when the A-domain is bound. Somewhat high B-factors in this region of the CopB ATPBD domain compared with the surrounding residues may reflect some structural flexibility.


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)

Secondary structure differences between the P-domains of CopB and Lp-CopA(A) Multiple sequence alignment of the residues corresponding to the loop/helical segment in the P-domains whose structures are known. The secondary structural elements are displayed for CopB (top) and Lp-CopA (bottom). Sequence alignment was performed using CLUSTALW [49]. The solvent accessible residues for CopB (top) and Lp-CopA (bottom) are indicated by bars running parallel to the alignment with colours ranging from white to dark blue. The accessibility information is extracted from the corresponding DSSP file. Blue indicates accessible, cyan indicates intermediate, and white indicates that the residues are buried. This Figure was prepared using the Program ESPript [50]. (B). Cartoon models of CopB (green) and Lp-CopA (blue) superimposed with the insert highlighting the different secondary structure elements between the two structures. The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera [48].
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Related In: Results  -  Collection

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Figure 2: Secondary structure differences between the P-domains of CopB and Lp-CopA(A) Multiple sequence alignment of the residues corresponding to the loop/helical segment in the P-domains whose structures are known. The secondary structural elements are displayed for CopB (top) and Lp-CopA (bottom). Sequence alignment was performed using CLUSTALW [49]. The solvent accessible residues for CopB (top) and Lp-CopA (bottom) are indicated by bars running parallel to the alignment with colours ranging from white to dark blue. The accessibility information is extracted from the corresponding DSSP file. Blue indicates accessible, cyan indicates intermediate, and white indicates that the residues are buried. This Figure was prepared using the Program ESPript [50]. (B). Cartoon models of CopB (green) and Lp-CopA (blue) superimposed with the insert highlighting the different secondary structure elements between the two structures. The structural alignment was performed using the structure comparison tool Matchmaker in the program UCSF Chimera. The ribbon diagram was generated using UCSF Chimera [48].
Mentions: In P-type ATPases, the P-domain interacts with the A-domain at different points in the catalytic cycle, as seen in the SERCA1 ATPase structures [17,42,43] and Lp-CopA structures [29]. The surface of the P-domain makes these interactions correspond to the region between β11 and β12 in the CopB structure. In SERCA1 and Lp-CopA (Figure 2), where interdomain contacts are seen, this region is helical. In the CopB ATPBD (Figure 2), as well as the ATPBDs of A. fulgidus CopA [28] and S. solfataricus CopB [24], this region has an irregular loop conformation. It is possible that domain–domain interactions influence the structure in this region of the P-domain, and a helical structure potentially could be adopted when the A-domain is bound. Somewhat high B-factors in this region of the CopB ATPBD domain compared with the surrounding residues may reflect some structural flexibility.

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