Structural and biophysical properties of the pathogenic SOD1 variant H46R/H48Q

Abstract

Over 100 mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Two pathogenic SOD1 mutations, His46Arg (H46R) and His48Gln (H48Q), affect residues that act as copper ligands in the wild type enzyme. Transgenic mice expressing a human SOD1 variant containing both mutations develop paralytic disease akin to ALS. Here we show that H46R/H48Q SOD1 possesses multiple characteristics that distinguish it from the wild type. These properties include the following: (1) an ablated copper-binding site, (2) a substantially weakened affinity for zinc, (3) a binding site for a calcium ion, (4) the ability to form stable heterocomplexes with the copper chaperone for SOD1 (CCS), and (5) compromised CCS-mediated oxidation of the intrasubunit disulfide bond in vivo. The results presented here, together with data on pathogenic SOD1 proteins coming from cell culture and transgenic mice, suggest that incomplete posttranslational modification of nascent SOD1 polypeptides via CCS may be a characteristic shared by familial ALS SOD1 mutants, leading to a population of destabilized, off-pathway folding intermediates that are toxic to motor neurons.

Figure 1. The structure of H46R/H48Q SOD1

A) The H46R/H48Q active site region in subunit A, which is similar to all subunits in both crystal forms. The zinc and electrostatic loop elements are colored orange and blue, respectively. The disulfide bond between C57 of the disulfide loop and C146 of the β-barrel is shown in yellow ball-and-stick. The zinc ion is shown as a green sphere. The R46 sidechain spans the active site channel and donates a hydrogen bond to a sidechain oxygen atom of D124 and the carbonyl oxygen of T137, both of the electrostatic loop. The Q48 sidechain displaces the guanidinium moiety of R143, preventing it from forming its normal hydrogen bonds to the carbonyl oxygen of C57. B) External zinc-binding site. The zinc and electrostatic loop elements are colored as in panel A). The sidechains of E77 and H110 from symmetry-related H46R/H48Q subunits coordinate zinc ions (green spheres) that bridge SOD1 dimers along the b-axis of the P21 crystal form. A zinc ion also occupies the zinc-binding site, capping the C-terminus of the small α-helix in the electrostatic loop. C) The calcium-binding site within each SOD1 monomer in the C222₁ structure (see text). The zinc and electrostatic loop elements are colored as in panels A) and B). The calcium ion (pink sphere) is held in a cleft between loop II (cyan) and the Greek key loop (loop VI, pale green) by the sidechain and carbonyl oxygen atoms of N26 and the carbonyl oxygen atoms of G27 and S102 in all but one subunit in the C222₁ crystal form. Water molecules acting as ligands to the calcium ion are shown as red spheres. D) 2FoFc (gray) and FoFc (blue) SIGMAA electron density for what appear to be diatomic covalent adducts on the Sγ atoms of C111 in the P2₁ structure. The 2FoFc and FoFc maps are contoured at 1 and 3 σ, respectively.

Figure 2. H46R/H48Q SOD1 has weakened affinity for zinc

A) Zinc Release Assay. In an effort to understand the molecular basis for the native gel-shift shown by the H46R/H48Q SOD1 double mutant in the presence of reducing agent (inset), the chelator 4-(2-Pyridylazo)-resorcinol (PAR) was used to monitor zinc release by H46R/H48Q SOD1 as shown by increased absorbance due to a newly formed PAR₂-Zinc complex. In the absence of reducing agent (middle trace), PAR slowly leaches zinc from the enzyme. In the presence of TCEP (top trace) zinc is released relatively rapidly from H46R/H48Q SOD1. Wild type SOD1 (lower trace) retains its copper and zinc ions under all conditions and even in the presence of 2 M GdnHCl on this timescale (30) (see text). B) Putative release of the zinc loop (loop IV, orange) from the β-barrel (pink) upon reduction of the intrasubunit disulfide bond as depicted in the wild type enzyme. The Sγ atoms of C57 and C146 are shown as yellow spheres, the copper ion is shown as a cyan sphere, and the zinc ion is shown as a green sphere. The electrostatic loop (loop VII) is shown in blue and is linked to the active site by hydrogen bonds between D124 and the nonliganding imidazole nitrogen atoms of copper ligand H46 and Zn ligand H71. H46R substitution is expected to impact H63, D124, and T137 weakening the interaction between the zinc and electrostatic loop elements as described in the text.

Figure 3. Non-denaturing gel electrophoresis of long-lived H46R/H48Q SOD1–CCS complexes

A) Wild-type yeast (left) and human (right) SOD1 proteins do not stably interact with their cognate copper chaperone proteins in non-denaturing PAGE of gel filtration chromatography (see Figure 4). B) A gel-shift characteristic of a 1:1 complex (circled) between H46R/H48Q SOD1 and yeast CCS (left) and human CCS (right) is observed, but only in the presence of reducing agent.

Figure 4. Isolation and characterization of the H46R/H48Q SOD1/CCS complex using analytical gel-filtration chromatography

A) The elution profile for a mixture of H46R/H48Q SOD1 and yeast CCS (gray trace) consists of two distinct peaks that emerge at a volume characteristic of a 54 kDa yCCS dimer and a slower eluting 32 kDa SOD1 dimer. The same 1:1 mixture of H46R/H48Q SOD1 in the presence of 10 mM TCEP (black trace) elutes from the column as a single peak characteristic of a 42 kDa SOD1/CCS heterodimer. B) The human form of CCS produces a similar elution profile to the yeast version when mixed with the SOD1 double mutant. Reducing agent must be present to induce complex formation.

Figure 5. Disulfide status of H46R/H48Q SOD1

The status of the intrasubunit disulfide bond in H46R/H48Q SOD1 coming from freshly lysed yeast and human embryonic kidney (HEK) cells probed using of 4-acetamide-4′-meleimidylstilbene-2, 2′-disulfunic acid (AMS), which modifies free thiols. The SOD1 monomer contains four cysteine residues, two of which make up a conserved disulfide bond. A) Yeast and B) HEK cells expressing H46R/H48Q SOD1 are lysed in the presence of AMS and the lysates are run on SDS-PAGE and probed with antibody specific for SOD1 as described (33, 34). In yeast, H46RH48Q SOD1 is expressed in ccs1-replete and ccs1Δ cells. In HEK cells, H46R/H48Q and hCCS are co-transfected as described previously (33). As indicated, AMS-modified thiols produce gel shifts to higher molecular weight such that two are modified in the disulfide oxidized enzyme and four are modified in the disulfide reduced enzyme. The in vivo disulfide status of the SOD1 double mutant whether from yeast cells or HEK cells remains predominantly reduced and the presence of CCS has no effect.

Figure 6. Positions of fALS associated SOD1 mutations and their postulated inhibitory effects on the SOD1 maturation pathway

The pink spheres represent mutations located within the β-barrel of SOD1. These mutations destabilize the metal-free disulfide-reduced (nascent) protein decreasing the fraction of molecules that are available for posttranslational modified by CCS. Unmodified nascent SOD1 proteins are either turned over or associate into soluble oligomeric complexes on the pathway to toxicity. The red spheres correspond to ALS mutations that adversely affect metal binding, the conformation of the zinc and electrostatic loop elements, and disulfide status. Although nascent pathogenic SOD1 mutants of this class are not generally destabilized relative to the nascent wild-type enzyme, full maturation cannot be achieved due to abrogated metal binding/coordination and/or disulfide oxidation, and these molecules remain off-pathway folding intermediates. The gold spheres correspond to ALS mutations that fall in the Greek key loop and might impact calcium ion binding and the stability of the nascent SOD1 protein.