TY - JOUR
T1 - Disease-associated mutations at copper ligand histidine residues of superoxide dismutase 1 diminish the binding of copper and compromise dimer stability
AU - Wang, Jiou
AU - Caruano-Yzermans, Amy
AU - Rodriguez, Angela
AU - Scheurmann, Jonathan P.
AU - Slunt, Hilda H.
AU - Cao, Xiaohang
AU - Gitlin, Jonathan
AU - Hart, P. John
AU - Borchelt, David R.
PY - 2007/1/5
Y1 - 2007/1/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33847003354&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33847003354&partnerID=8YFLogxK
U2 - 10.1074/jbc.M604503200
DO - 10.1074/jbc.M604503200
M3 - Article
C2 - 17092942
AN - SCOPUS:33847003354
SN - 0021-9258
VL - 282
SP - 345
EP - 352
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 1
ER -