Copper serves as a cofactor for many enzymes involved in important biological processes, but can also facilitate the formation of toxic organic and oxygen radicals. A host of proteins, including membrane transporters, metallochaperones, and metalloregulatory proteins, maintains intracellular copper concentrations such that copper ions are provided to essential enzymes, but do not accumulate to deleterious levels. Understanding how these proteins function on the molecular level is the theme of this ongoing research program. Despite significant progress toward determining how soluble copper trafficking proteins bind metal ions, recognize physiological partners, and facilitate metal ion transfer, there are large gaps in the current understanding of copper homeostasis, particularly regarding copper translocation across membranes by P1B- type ATPases. Importantly, mutations in human Cu+transporting P1B-type ATPases lead to Wilson disease and Menkes syndrome, serious disorders of copper metabolism. To complete the molecular picture of copper trafficking and to advance understanding of Cu+ATPases, a model system from the hyperthermophile Archaeoglobus fulgidus is being investigated. The A. fulgidus CopA Cu+ATPase contains all of the structural elements that are present in the human Wilson and Menkes disease proteins, including soluble metal binding domains (MBDs), an ATP binding domain (ATPBD), and an actuator domain (A-domain). Other components of the A. fulgidus pathway include a novel CopZ copper chaperone and a putative transcriptional regulator, CopT. The proposed research involves biophysical and structural characterization of CopA, CopZ, and CopT. The soluble CopA MBDs will be structurally characterized and protein-protein interactions between the MBDs and the CopZ chaperone will be investigated. The copper binding properties, potential interactions with CopZ, and structure of CopT will be probed. Finally, state-of-the-art crystallization techniques for membrane proteins will be applied to CopA, and structures determined in multiple conformations. These data will provide molecular insight into the molecular basis for Wilson and Menkes diseases as well as adding to the database of membrane protein structures. A number of human diseases are linked to deficiencies in cellular handling of copper, which is an essential yet potentially toxic metal ion. This project will provide a molecular picture of the type of protein that is defective in Wilson disease and Menkes syndrome, both genetic disorders of copper metabolism. These same proteins may also be associated with resistance to anticancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058518-12
Application #
7758730
Study Section
Special Emphasis Panel (ZRG1-BCMB-B (02))
Program Officer
Smith, Ward
Project Start
1999-02-01
Project End
2012-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
12
Fiscal Year
2010
Total Cost
$300,700
Indirect Cost
Name
Northwestern University at Chicago
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
160079455
City
Evanston
State
IL
Country
United States
Zip Code
60201
Ross, Matthew O; Rosenzweig, Amy C (2017) A tale of two methane monooxygenases. J Biol Inorg Chem 22:307-319
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Lawton, Thomas J; Rosenzweig, Amy C (2016) Biocatalysts for methane conversion: big progress on breaking a small substrate. Curr Opin Chem Biol 35:142-149
Lawton, Thomas J; Kenney, Grace E; Hurley, Joseph D et al. (2016) The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins. Biochemistry 55:2278-90
Lawton, Thomas J; Rosenzweig, Amy C (2016) Methane-Oxidizing Enzymes: An Upstream Problem in Biological Gas-to-Liquids Conversion. J Am Chem Soc 138:9327-40
Smith, Aaron T; Barupala, Dulmini; Stemmler, Timothy L et al. (2015) A new metal binding domain involved in cadmium, cobalt and zinc transport. Nat Chem Biol 11:678-84
Kathman, Stefan G; Span, Ingrid; Smith, Aaron T et al. (2015) A Small Molecule That Switches a Ubiquitin Ligase From a Processive to a Distributive Enzymatic Mechanism. J Am Chem Soc 137:12442-5
Smith, Aaron T; Smith, Kyle P; Rosenzweig, Amy C (2014) Diversity of the metal-transporting P1B-type ATPases. J Biol Inorg Chem 19:947-60
Chang, Wei-chen; Guo, Yisong; Wang, Chen et al. (2014) Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics. Science 343:1140-4
Makhlynets, Olga; Boal, Amie K; Rhodes, Delacy V et al. (2014) Streptococcus sanguinis class Ib ribonucleotide reductase: high activity with both iron and manganese cofactors and structural insights. J Biol Chem 289:6259-72

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