Abstract
The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.
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Acknowledgments
We are grateful to Jürgen Schlitter and Steffen Wolff for stimulating discussions and Ken Sale at Sandia National Laboratories for molecular modeling advice. This work was supported by grant 3100A0-109703 from the Swiss National Foundation (MS), a grant from the International Copper Association (MS), grant I/78128 from the VolkswagenStiftung (ML), a grant by the Deutsche Forschungsgemeinschaft LU405/3-1 (ML), and by the Laboratory Directed Research and Development program at Sandia National Laboratories, which is a multi-program laboratory operated by Sandia Corporation, a Lockheed Martin Company for the United States Department of Energy under contract DE-AC04-94AL85000. R.S. gratefully acknowledges generous support from the DFG (SFB 642). G.K. is a fellow of the RUB Research School.
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Lübben, M., Portmann, R., Kock, G. et al. Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking. Biometals 22, 363–375 (2009). https://doi.org/10.1007/s10534-008-9173-4
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DOI: https://doi.org/10.1007/s10534-008-9173-4