Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking |
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Authors: | Mathias Lübben Reto Portmann Gerd Kock Raphael Stoll Malin M Young Marc Solioz |
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Institution: | (1) Department of Biophysics, Ruhr-University Bochum, 44780 Bochum, Germany;(2) Department of Clinical Pharmacology and Visceral Research, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland;(3) Department of Biochemistry II, Biomolecular NMR, Ruhr-University Bochum, 44780 Bochum, Germany;(4) Sandia National Laboratory, Livermore, CA, USA |
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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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Keywords: | Copper ATPase Structure model Cross-linking Mass spectrometry Serca1 |
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