P. John Hart

Misfolded CuZnSOD and amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of motor neurons. The inherited form of the disease, familial ALS, represents 5–10% of the total cases, and the best documented of these are due to lesions in SOD1, the gene encoding copper–zinc superoxide dismutase (CuZnSOD). The mechanism by which mutations in SOD1 cause familial ALS is currently unknown. Two hypotheses have dominated recent discussion of the toxicity of ALS mutant CuZnSOD proteins: the oligomerization hypothesis and the oxidative damage hypothesis. The oligomerization hypothesis maintains that mutant CuZnSOD proteins are, or become, misfolded and consequently oligomerize into increasingly high-molecular-weight species that ultimately lead to the death of motor neurons. The oxidative damage hypothesis maintains that ALS mutant CuZnSOD proteins catalyze oxidative reactions that damage substrates critical for viability of the affected cells. This perspective reviews some of the properties of both wild-type and mutant CuZnSOD proteins, suggests how these properties may be relevant to these two hypotheses, and proposes that these two hypotheses are not necessarily mutually exclusive.

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.

Molecular Recognition of Insulin by a Synthetic Receptor

The discovery of molecules that bind tightly and selectively to desired proteins continues to drive innovation at the interface of chemistry and biology. This paper describes the binding of human insulin by the synthetic receptor cucurbit[7]uril (Q7) in vitro. Isothermal titration calorimetry and fluorescence spectroscopy experiments show that Q7 binds to insulin with an equilibrium association constant of 1.5 × 10(6) M(-1) and with 50-100-fold selectivity versus proteins that are much larger but lack an N-terminal aromatic residue, and with >1000-fold selectivity versus an insulin variant lacking the N-terminal phenylalanine (Phe) residue. The crystal structure of the Q7·insulin complex shows that binding occurs at the N-terminal Phe residue and that the N-terminus unfolds to enable binding. These findings suggest that site-selective recognition is based on the properties inherent to a protein terminus, including the unique chemical epitope presented by the terminal residue and the greater freedom of the terminus to unfold, like the end of a ball of string, to accommodate binding. Insulin recognition was predicted accurately from studies on short peptides and exemplifies an approach to protein recognition by targeting the terminus.

Variation in aggregation propensities among ALS-associated variants of SOD1: Correlation to human disease

To date, 146 different mutations in superoxide dismutase 1 (SOD1) have been identified in patients with familial amyotrophic lateral sclerosis (ALS). The mean age of disease onset in patients inheriting mutations in SOD1 is 45–47 years of age. However, although the length of disease duration is highly variable, there are examples of consistent disease durations associated with specific mutations (e. g. A4V, less than 2 years). In the present study, we have used a large set of data from SOD1-associated ALS pedigrees to identify correlations between disease features and biochemical/biophysical properties of more than 30 different variants of mutant SOD1. Using a reliable cell culture assay, we show that all ALS-associated mutations in SOD1 increase the inherent aggregation propensity of the protein. However, the relative propensity to do so varied considerably among mutants. We were not able to explain the variation in aggregation rates by differences in known protein properties such as enzyme activity, protein thermostability, mutation position or degree of change in protein charge. Similarly, we were not able to explain variability in the duration of disease in SOD1-associated ALS pedigrees by these properties. However, we find that the majority of pedigrees in which patients exhibit reproducibly short disease durations are associated with mutations that show a high inherent propensity to induce aggregation of SOD1.

The copper-iron connection in biology : structure of the metallo-oxidase Fet3p

Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(II) to Fe(III). This ferrous iron oxidation is coupled to the reduction of O2 to H2O and is termed the ferroxidase reaction. Fet3p-produced Fe(III) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(II) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase.

Structure of the Hsp110:Hsc70 Nucleotide Exchange Machine

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner proteins NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.

Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS

Transgenic mice that model familial (f)ALS, caused by mutations in superoxide dismutase (SOD)1, develop paralysis with pathology that includes the accumulation of aggregated forms of the mutant protein. Using a highly sensitive detergent extraction assay, we traced the appearance and abundance of detergent-insoluble and disulfide cross-linked aggregates of SOD1 throughout the disease course of SOD1-fALS mice (G93A, G37R, and H46R/H48Q). We demonstrate that the accumulation of disulfide cross-linked, detergent-insoluble, aggregates of mutant SOD1 occurs primarily in the later stages of the disease, concurrent with the appearance of rapidly progressing symptoms. We find no evidence for a model in which aberrant intermolecular disulfide bonding has an important role in promoting the aggregation of mutant SOD1, instead, such cross-linking appears to be a secondary event. Also, using both cell culture and mouse models, we find that mutant protein lacking the normal intramolecular disulfide bond is a major component of the insoluble SOD1 aggregates. Overall, our findings suggest a model in which soluble forms of mutant SOD1 initiate disease with larger aggregates implicated only in rapidly progressing events in the final stages of disease. Within the final stages of disease, abnormalities in the oxidation of a normal intramolecular disulfide bond in mutant SOD1 facilitate the aggregation of mutant protein.

The refined crystal structure of an endochitinasefrom Hordeum vulgare L. seeds at 1.8 Å resolution

Class II chitinases (EC 3.2.1.14) are plant defense proteins. They hydrolyze chitin, an insoluble beta-1,4-linked polymer of N-acetylglucosamine (NAG), which is a major cell-wall component of many fungal hyphae. We previously reported the three-dimensional structure of the 26 kDa class II endochitinase from barley seeds at 2.8 A resolution, determined using multiple isomorphous replacement (MIR) methods. Here, we report the crystallographic refinement of this chitinase structure against data to 1.8 A resolution using rounds of hand rebuilding coupled with molecular dynamics (X-PLOR). The final model has an R-value of 18.1% for the 5.0 to 1.8 A data shell and 19.8% for the 10.0 to 1.8 A shell, and root-mean-square deviations from standard bond lengths and angles of 0.017 A and 2.88 degrees, respectively. The 243 residue molecule has one beta-sheet, ten alpha-helices and three disulfide bonds; 129 water molecules are included in the final model. We show structural comparisons confirming that chitinase secondary structure resembles lysozyme at the active site region. Based on substrate binding to lysozyme, we have built a hypothetical model for the binding of a hexasaccharide into the pronounced active site cleft of chitinase. This provides the first view of likely substrate interactions from this family of enzymes; the model is consistent with a lysozyme-like mechanism of action in which Glu67 acts as proton donor and Glu89 is likely to stabilize the transition state oxycarbonium ion. These binding site residues, and many hydrophobic residues are conserved in a range of plant chitinases. This endochitinase structure will serve as a model for other plant chitinases, and that catalytic models based on this structure will be applicable to the entire enzyme family.

Immature Copper-Zinc Superoxide Dismutase and Familial Amyotrophic Lateral Sclerosis

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease, motor neuron disease). Insoluble forms of mutant SOD1 accumulate in neural tissues of human ALS patients and in spinal cords of transgenic mice expressing these polypeptides, suggesting that SOD1-linked ALS is a protein misfolding disorder. Understanding the molecular basis for how the pathogenic mutations give rise to SOD1 folding intermediates, which may themselves be toxic, is therefore of keen interest. A critical step on the SOD1 folding pathway occurs when the copper chaperone for SOD1 (CCS) modifies the nascent SOD1 polypeptide by inserting the catalytic copper cofactor and oxidizing its intrasubunit disulfide bond. Recent studies reveal that pathogenic SOD1 proteins coming from cultured cells and from the spinal cords of transgenic mice tend to be metal-deficient and/or lacking the disulfide bond, raising the possibility that the disease-causing mutations may enhance levels of SOD1-folding intermediates by preventing or hindering CCS-mediated SOD1 maturation. This mini-review explores this hypothesis by highlighting the structural and biophysical properties of the pathogenic SOD1 mutants in the context of what is currently known about CCS structure and action. Other hypotheses as to the nature of toxicity inherent in pathogenic SOD1 proteins are not covered.

Structure of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A in Complex with Protein-Bound Methionine

Peptide methionine sulfoxide reductase (MsrA) repairs oxidative damage to methionine residues arising from reactive oxygen species and reactive nitrogen intermediates. MsrA activity is found in a wide variety of organisms, and it is implicated as one of the primary defenses against oxidative stress. Disruption of the gene encoding MsrA in several pathogenic bacteria responsible for infections in humans results in the loss of their ability to colonize host cells. Here, we present the X-ray crystal structure of MsrA from the pathogenic bacterium Mycobacterium tuberculosis refined to 1.5 A resolution. In contrast to the three catalytic cysteine residues found in previously characterized MsrA structures, M. tuberculosis MsrA represents a class containing only two functional cysteine residues. The structure reveals a methionine residue of one MsrA molecule bound at the active site of a neighboring molecule in the crystal lattice and thus serves as an excellent model for protein-bound methionine sulfoxide recognition and repair.