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J. Biol. Chem., Vol. 282, Issue 1, 345-352, January 5, 2007

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Disease-associated Mutations at Copper Ligand Histidine Residues of Superoxide Dismutase 1 Diminish the Binding of Copper and Compromise Dimer Stability^*

Jiou Wang¹, Amy Caruano-Yzermans¹, Angela Rodriguez^¶1, Jonathan P. Scheurmann^¶1, Hilda H. Slunt^||1, Xiaohang Cao^¶, Jonathan Gitlin, P. John Hart^¶**, and David R. Borchelt^||2

From the Departments of Pathology and Neuroscience, Johns Hopkins University, Baltimore, Maryland 21205, the Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, the ^¶Department of Biochemistry, the ^**X-ray Crystallography Core Laboratory, and the Geriatric Research, Education, and Clinical Center, Department of Veteran's Affairs, South Texas Veterans Health Care System, The University of Texas San Antonio Health Sciences Center, San Antonio, Texas 78229, and the ^||Department of Neuroscience, Santa Fe Health Alzheimer's Disease Research Center, McKnight Brain Institute, University of Florida, Gainesville, Florida 32611

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with ^-ONOO and H₂O₂ in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind copper. We find that single mutations at copper ligands, including H46R, H48Q, and a quadruple mutant H46R/H48Q/H63G/H120G, also diminish the binding of radioactive copper. Further, using native polyacrylamide gel electrophoresis and a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define disease-causing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry.

Received for publication, May 10, 2006 , and in revised form, August 31, 2006.

The atomic coordinates and structure factors (code 2NNX) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health (NIH) Grant NIH44464 (to J. G.), by the ALS Association (to D. R. B.), by the Muscular Dystrophy Association (to D. R. B.), by Robert A. Welch Foundation Grant AQ-1399 (to P. J. H.), by the Packard Center for ALS Research at JHU (to D. R. B.), and by NINDS, NIH Grants P01 NS049134 (to D. R. B. and P. J. H.) and R01 NS39912 (to P. J. H.). The production of ⁶⁴Cu at the Washington University School of Medicine is supported by NCI, NIH Grant R24 CA86307. The X-ray Crystallography Core Laboratory is supported by the VPR and the Executive Research Council at the University of Texas Health Science Center at San Antonio. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, references, and Figs. S1 and S2.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Neuroscience, University of Florida, 100 Newell Drive, Room L1-100H, P. O. Box 100244, Gainesville, FL 32610-0244. Tel.: 352-294-0105; Fax: 352-392-8347; E-mail: Borchelt{at}mbi.ufl.edu.

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