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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 diagram of the apo-ATPBD of A. fulgidus CopBThe highly conserved residues in the P- and N-domains are highlighted as sticks. The residues Asp389, Gly392 and Thr393 form Motif I, whereas Thr537 forms Motif II, and Lys565, Asp583 and Asp587 form Motif III of the HAD super family. The secondary structural elements (α1–α10 and β1–β12) are annotated alongside the respective α-helices and β-strands. Ribbon diagrams were generated using UCSF Chimera [48].
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Figure 1: Ribbon diagram of the apo-ATPBD of A. fulgidus CopBThe highly conserved residues in the P- and N-domains are highlighted as sticks. The residues Asp389, Gly392 and Thr393 form Motif I, whereas Thr537 forms Motif II, and Lys565, Asp583 and Asp587 form Motif III of the HAD super family. The secondary structural elements (α1–α10 and β1–β12) are annotated alongside the respective α-helices and β-strands. Ribbon diagrams were generated using UCSF Chimera [48].

Mentions: The 1.6 Å resolution crystal structure of the ATPBD of A. fulgidus CopB was determined using MAD phasing from a selenomethionine derivative. Similar to the structures of ATPBDs of other known P-type ATPases, the A. fulgidus CopB ATPBD has a kidney bean-like topology in which the P- and N-domains are separated by two short loops (Figure 1). The CopB ATPBD is structurally very similar to the ATPBD of CopA from the same organism (PDB code 2B8E [25]) with an overall RMSD (root mean square deviation) of 0.9 Å. Structural comparisons of the CopB ATPBD with the ATPBDs of other well-characterized ATPases, such as Na+, K+, SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1), WND (Wilson disease protein) and Kdp, reveal that the archaeal ATPBD has fewer helices, strands and loops than the eukaryotic ATPBDs, particularly in the N-domain [17,27,38]. The eukaryotic ATPBDs possess several more secondary structural elements in addition to longer loop regions (10–50 residues long) [17,27,38]. These differences are apparent in the TOPS topology diagrams comparing the eukaryotic Ca(II) transporter SERCA1 and CopB ATPBDs (Supplementary Figure S2 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm).


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 diagram of the apo-ATPBD of A. fulgidus CopBThe highly conserved residues in the P- and N-domains are highlighted as sticks. The residues Asp389, Gly392 and Thr393 form Motif I, whereas Thr537 forms Motif II, and Lys565, Asp583 and Asp587 form Motif III of the HAD super family. The secondary structural elements (α1–α10 and β1–β12) are annotated alongside the respective α-helices and β-strands. Ribbon diagrams were generated using UCSF Chimera [48].
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Related In: Results  -  Collection

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Figure 1: Ribbon diagram of the apo-ATPBD of A. fulgidus CopBThe highly conserved residues in the P- and N-domains are highlighted as sticks. The residues Asp389, Gly392 and Thr393 form Motif I, whereas Thr537 forms Motif II, and Lys565, Asp583 and Asp587 form Motif III of the HAD super family. The secondary structural elements (α1–α10 and β1–β12) are annotated alongside the respective α-helices and β-strands. Ribbon diagrams were generated using UCSF Chimera [48].
Mentions: The 1.6 Å resolution crystal structure of the ATPBD of A. fulgidus CopB was determined using MAD phasing from a selenomethionine derivative. Similar to the structures of ATPBDs of other known P-type ATPases, the A. fulgidus CopB ATPBD has a kidney bean-like topology in which the P- and N-domains are separated by two short loops (Figure 1). The CopB ATPBD is structurally very similar to the ATPBD of CopA from the same organism (PDB code 2B8E [25]) with an overall RMSD (root mean square deviation) of 0.9 Å. Structural comparisons of the CopB ATPBD with the ATPBDs of other well-characterized ATPases, such as Na+, K+, SERCA1 (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1), WND (Wilson disease protein) and Kdp, reveal that the archaeal ATPBD has fewer helices, strands and loops than the eukaryotic ATPBDs, particularly in the N-domain [17,27,38]. The eukaryotic ATPBDs possess several more secondary structural elements in addition to longer loop regions (10–50 residues long) [17,27,38]. These differences are apparent in the TOPS topology diagrams comparing the eukaryotic Ca(II) transporter SERCA1 and CopB ATPBDs (Supplementary Figure S2 at http://www.bioscirep.org/bsr/032/bsr0320443add.htm).

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