Richard W. Strange

Amyloid-Like Filaments and Water-Filled Nanotubes Formed by Sod1 Mutant Proteins Linked to Familial Als

Mutations in the SOD1 gene cause the autosomal dominant, neurodegenerative disorder familial amyotrophic lateral sclerosis (FALS). In spinal cord neurons of human FALS patients and in transgenic mice expressing these mutant proteins, aggregates containing FALS SOD1 are observed. Accumulation of SOD1 aggregates is believed to interfere with axonal transport, protein degradation and anti-apoptotic functions of the neuronal cellular machinery. Here we show that metal-deficient, pathogenic SOD1 mutant proteins crystallize in three different crystal forms, all of which reveal higher-order assemblies of aligned β-sheets. Amyloid-like filaments and water-filled nanotubes arise through extensive interactions between loop and β-barrel elements of neighboring mutant SOD1 molecules. In all cases, non-native conformational changes permit a gain of interaction between dimers that leads to higher-order arrays. Normal β-sheet–containing proteins avoid such self-association by preventing their edge strands from making intermolecular interactions. Loss of this protection through conformational rearrangement in the metal-deficient enzyme could be a toxic property common to mutants of SOD1 linked to FALS.

Crystal Structure of Human Prion Protein Bound to a Therapeutic Antibody.

Prion infection is characterized by the conversion of host cellular prion protein (PrPC) into disease-related conformers (PrPSc) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrPC rather than PrPSc. We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrPC and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrPC conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular β-sheet. The importance of this residue in mediating protein–protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.

The Structure of Human Extracellular Copper–Zinc Superoxide Dismutase at 1.7 Å Resolution: Insights into Heparin and Collagen Binding

Extracellular superoxide dismutase (SOD3) is a homotetrameric copper- and zinc-containing glycoprotein with affinity for heparin. The level of SOD3 is particularly high in blood vessel walls and in the lungs. The enzyme has multiple roles including protection of the lungs against hyperoxia and preservation of nitric oxide. The common mutation R213G, which reduces the heparin affinity of SOD3, is associated with increased risk of myocardial infarctions and stroke. We report the first crystal structure of human SOD3 at 1.7 A resolution. The overall subunit fold and the subunit-subunit interface of the SOD3 dimer are similar to the corresponding structures in Cu-Zn SOD (SOD1). The metal-binding sites are similar to those found in SOD1, but with Asn180 replacing Thr137 at the Cu-binding site and a much shorter loop at the zinc-binding site. The dimers form a functional homotetramer that is fashioned through contacts between two extended loops on each subunit. The N- and C-terminal end regions required for tetramerisation and heparin binding, respectively, are highly flexible. Two grooves fashioned by the tetramer interface are suggestive as the probable sites for heparin and collagen binding.

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

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.

A critical assessment of the evidence from XAFS and crystallography for the breakage of the imidazolate bridge during catalysis in CuZn superoxide dismutase

BACKGROUND Copper-zinc superoxide dismutase (CuZn SOD) protects cells from the toxic effects of superoxide radicals. Key steps in the catalytic mechanism of CuZn SOD are thought to be the breakage of the imidazolate bridge between copper and zinc upon reduction of the copper site and the subsequent proton donation from the bridging histidine. This view has been recently challenged by a crystallographic study at 1.9 A resolution where evidence for a five-coordinate copper site in the reduced enzyme was provided. In contrast, a crystallographic study of yeast CuZn SOD at 1.7 A has confirmed the breaking of the bridging histidine in reduced crystals. We have examined the nature of the changes in metal coordination which result upon reduction of the enzyme using the X-ray absorption fine structure (XAFS) technique. RESULTS The copper and zinc K-edge XAFS data of bovine SOD, recorded in the buffer systems used in the two crystallographic studies, were analyzed by constrained refinement using fast curved wave theory, taking full account of multiple-scattering effects. The study confirms that in the oxidized form of the enzyme the copper atom is five coordinate, with four histidine ligands at 1.99 +/- 0.02 A and a water molecule at 2.18 +/- 0.03 A. In the reduced form of the enzyme, one of the histidine ligands and the water molecule are lost from the inner coordination sphere of the copper, with the three remaining histidines ligated at 1.97 +/- 0.02 A. The X-ray absorption near edge structure (XANES) of the reduced enzyme is consistent with an approximate trigonal planar geometry at the copper site. The XAFS at the zinc K-edge is essentially identical in both the oxidized and reduced enzyme and is accounted for by three histidines coordinated at 2.01 +/- 0.02 A and an aspartate ligand at 1.96 +/- 0.03 A. CONCLUSIONS The existence of a three-coordinate cuprous ion in bovine CuZn SOD is demonstrated and is a key feature of catalytic degradation of superoxide substrate by SOD involving alternate Cu(I) and Cu(II) states of the enzyme. Only subtle changes in the zinc K-edge XAFS take place upon reduction. Thus the reaction mechanism which involves breakage of the bridging histidine is unambiguously supported by the XAFS data.

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg

The His46Arg (H46R) mutant of human copper‐zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn‐loaded (Zn‐H46R) and metal‐free (apo‐H46R) forms. The Zn‐H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 “secondary bridge” between the copper‐ and zinc‐binding sites is disrupted, and the “electrostatic loop” and “zinc loop” elements are largely disordered. The apo‐H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1–SOD1 interactions lead to the formation of higher‐order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.

Molecular Dynamics Using Atomic-Resolution Structure Reveal Structural Fluctuations that May Lead to Polymerization of Human Cu-Zn Superoxide Dismutase.

Mutations of the gene encoding Cu–Zn superoxide dismutase (SOD1) cause 20% of the familial cases of the progressive neurodegenerative disease ALS. A growing body of evidence suggests that in familial ALS (FALS) it is the molecular behavior of the metal-depleted SOD1 dimer that leads to a gain of toxic properties by misfolding, unfolding, and aggregation. Structural studies have so far provided static snapshots on the behavior of the wild-type enzyme and some of the FALS mutants. New approaches are required to map out the structural trajectories of the molecule. Here, using our 1.15-Å resolution structure of fully metallated human SOD1 and highly parallelized molecular dynamics code on a high-performance capability computer, we have undertaken molecular dynamics calculations to 4,000 ps to reveal the first stages of misfolding caused by metal deletion. Large spatial and temporal fluctuations of the “electrostatic” and “Zn-binding” loops adjacent to the metal-binding sites are observed in the apo-enzyme relative to the fully metallated dimer. These early misfolding events expose the β-barrels of the dimer to the external environment, allowing close interactions with adjacent molecules. Protection of the β-edge of the protein can be partially restored by incorporating a single Zn molecule per dimer. These calculations reveal an essential step in the formation of the experimentally observed self-aggregations of metal-depleted FALS mutant SOD1. This result also has implications for the role of demetallated wild-type SOD1 in sporadic cases of ALS, for which the molecular cause still remains undiscovered.

Structural and biophysical properties of metal-free pathogenic SOD1 mutants A4V and G93A.

Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS. The A4V variant is responsible for the majority of familial ALS cases in North America, causing rapidly progressing paralysis once symptoms begin and the G93A SOD1 variant is overexpressed in often studied murine models of the disease. Here we report the three-dimensional structures of metal-free A4V and of metal-bound and metal-free G93A SOD1. In the metal-free structures, the metal-binding loop elements are observed to be severely disordered, suggesting that these variants may share mechanisms of aggregation proposed previously for other pathogenic SOD1 proteins.

The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase.

Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding. X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre. The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu–histidine bond by approximately 0.08 Å. Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy

X-ray absorption spectroscopy is exquisitely sensitive to the coordination geometry of an absorbing atom and therefore allows bond distances and angles of the surrounding atomic cluster to be measured with atomic resolution. By contrast, the accuracy and resolution of metalloprotein active sites obtainable from x-ray crystallography are often insufficient to analyze the electronic properties of the metals that are essential for their biological functions. Here, we demonstrate that the combination of both methods on the same metalloprotein single crystal yields a structural model of the protein with exceptional active-site resolution. To this end, we have collected an x-ray diffraction data set to 1.4-Å resolution and Fe K-edge polarized x-ray absorption near edge structure (XANES) spectra on the same cyanomet sperm whale myoglobin crystal. The XANES spectra were quantitatively analyzed by using a method based on the multiple scattering approach, which yielded Fe-heme structural parameters with ±(0.02–0.07)-Å accuracy on the atomic distances and ±7° on the Fe–CN angle. These XANES-derived parameters were subsequently used as restraints in the crystal structure refinement. By combining XANES and x-ray diffraction, we have obtained an cyanomet sperm whale myoglobin structural model with a higher precision of the bond lengths and angles at the active site than would have been possible with crystallographic analysis alone.