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

Abstract

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with ؊ ONOO and H 2 O 2 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 diseasecausing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry. Amyotrophic lateral sclerosis (ALS),3 which is characterized by progressive muscle weakness and motor neuron loss, presents as both sporadic and familial (FALS) illness. A subset of FALS cases is caused by missense mutations in the superoxide scavenging enzyme, Cu/Zn-superoxide dismutase 1 (SOD1) (1, 2). To date, over 100 different point mutations, and Ͼ5 early termination mutations have been linked to FALS (www. alsod.org) (for reviews see Refs. 2-4). Early studies of FALS-SOD1 enzymes demonstrated that some mutants retain high levels of activity and relatively long half lives (5). Moreover, mutant proteins that are inactive or short-lived do not exhibit evidence of dominant negative action with regard to the superoxide-scavenging activity of enzyme derived from the normal allele (6). In transgenic mice, the hyperexpression of the G93A and G37R variants of FALS-SOD1 increases superoxide scavenging activity, kills motor neurons, and causes paralysis (7,8). SOD1 knock-out mice do not develop ALS-like phenotypes but do show sensory and motor neuropathy (9). Together, these studies establish that SOD1 mutations cause ALS through a gained toxic property. Although expressed ubiquitously (8, 10), mutant SOD1 selectively damages motor neurons, by mechanisms yet to be fully understood.

A Common Property of Amyotrophic Lateral Sclerosis-associated Variants

“…3) are also consistent with the existing hypothesis that fALS variants may perturb copper-mediated chemistry (7,59), because the electrostatic loop contains copper ligands and normally prohibits access of small molecules to the active site. The finding that proteins that do not bind active site copper still cause ALS, however, argues against copper-mediated chemistry as a universal gain of toxic SOD1 function (39,58,60).…”

“…A number of lines of evidence support a role for metal deficiency in ALS, including: 1) five of the variants studied, G85R, S134N, D14V, D125H, and H46R, the so called metal-binding site variants, are metal-deficient in vivo (25,54); 2) copperdeficient SOD1 is of potential physiological relevance, considering that roughly 35% of human copper/zinc SOD1 is copperdeficient in vivo (55)(56)(57); and 3) copper-deficient SOD1 can cause fALS in mice (39,58). Our results demonstrate that zinc deficiency, more so than copper deficiency, is related to disorder in the electrostatic loop (Fig.…”

Structures of the G85R Variant of SOD1 in Familial Amyotrophic Lateral Sclerosis

“…3, His 110 from one G85R dimer and Glu 24 from another together with two thiocyanate anions coordinate zinc in a tetrahedral geometry, creating tightly linked G85R SOD1 layers. Although zinc has not been observed to bind at this location in any wild type SOD1 structure determined to date, zinc is observed to bridge dimers through His 110 and Glu 77 in the crystal structure of the H46R/H48Q double copper site SOD1 variant, which contains an ablated copper-binding site (Protein Data Bank code 2NNX (44) …”

Metal Deficiency Increases Aberrant Hydrophobicity of Mutant Superoxide Dismutases That Cause Amyotrophic Lateral Sclerosis

“…However, it has not been shown that zincdeficient, copper-replete SOD1 is produced in vivo as a consequence of ALS mutations, and loading of copper into SOD1 by the copper chaperone for SOD1 (CCS) is not required for toxicity (34,35). Furthermore, the MBR mutants have a disrupted copper site and have been found to be severely deficient in both zinc and copper (17,30), yet expression of these SOD1s still produces motor neuron disease (1,2,30,34,36,37).…”