No abstract

Copper chaperone for superoxide dismutase 1 (SOD1), CCS, is the physiological partner for the complex mechanism of SOD1 maturation. We report an in vitro model for human CCS-dependent SOD1 maturation based on the study of the interactions of human SOD1 (hSOD1) with full-length WT human CCS (hCCS), as well as with hCCS mutants and various truncated constructs comprising one or two of the protein’s three domains. The synergy between electrospray ionization mass spectrometry (ESI-MS) and NMR is fully exploited. This is an in vitro study of this process at the molecular level. Domain 1 of hCCS is necessary to load hSOD1 with Cu(I), requiring the heterodimeric complex formation with hSOD1 fostered by the interaction with domain 2. Domain 3 is responsible for the catalytic formation of the hSOD1 Cys-57–Cys-146 disulfide bond, which involves both hCCS Cys-244 and Cys-246 via disulfide transfer.

Mutations in the superoxide dismutase 1 (SOD1) gene are related to familial cases of amyotrophic lateral sclerosis (fALS). Here we exploit in-cell NMR to characterize the protein folding and maturation of a series of fALS-linked SOD1 mutants in human cells and to obtain insight into their behaviour in the cellular context, at the molecular level. The effect of various mutations on SOD1 maturation are investigated by changing the availability of metal ions in the cells, and by coexpressing the copper chaperone for SOD1, hCCS. We observe for most of the mutants the occurrence of an unstructured SOD1 species, unable to bind zinc. This species may be a common precursor of potentially toxic oligomeric species, that are associated with fALS. Coexpression of hCCS in the presence of copper restores the correct maturation of the SOD1 mutants and prevents the formation of the unstructured species, confirming that hCCS also acts as a molecular chaperone.

Cellular acquisition of copper in eukaryotic organisms is primarily accomplished through high-affinity copper transport proteins (Ctr). The extracellular N-terminal regions of both human and yeast Ctr1 contain multiple methionine residues organized in copper-binding Mets motifs. These motifs comprise combinations of methionine residues arranged in clusters of MXM and MXXM, where X can be one of several amino acids. Model peptides corresponding to 15 different Mets motifs were synthesized and determined to selectively bind Cu(I) and Ag(I), with no discernible affinity for divalent metal ions. These are rare examples of biological thioether-only metal binding sites. Effective dissociation constant (KD) values for the model Mets peptides and Cu(I) were determined by an ascorbic acid oxidation assay and validated through electrospray ionization mass spectrometry and range between 2 and 11 microM. Affinity appears to be independent of pH, the arrangement of the motif, and the composition of intervening amino acids, all of which reveal the generality and flexibility of the MX1-2MX1-2M domain. Circular dichroism spectroscopy, 1H-NMR spectroscopy, and X-ray absorption spectroscopy were also used to characterize the binding event. These results are intended to aid the development of the still unknown mechanism of copper transport across the cell membrane.

The N-terminal, extracellular regions of eukaryotic high affinity copper transport (Ctr) proteins vary in composition of the Cu(i) binding amino acids: methionine, histidine, and cysteine. To examine why certain amino acids are exploited over others in Ctrs from different organisms, the relative Cu(i) binding affinity and the dependence of binding on pH were examined for 3 peptides of the sequence MG(2)XG(2)MK, where X is either Met, His, or Cys. Cu(i) affinity was examined using an ascorbic acid oxidation assay, an electrospray ionization mass spectrometry technique, and spectrophotometric titration with a competitive Cu(i) chelator. The relative affinities of the peptides with Cu(i) reveal a trend whereby Cys > His > Met at pH 7.4 and Cys > Met > His at pH 4.5. Ligand geometry and metric parameters were determined with X-ray absorption spectroscopy. Susceptibility of the peptides to oxidation by hydrogen peroxide and copper-catalyzed oxidative conditions was evaluated by mass spectrometry. These results support hypotheses as to why certain Cu(i) binding amino acids are preferred over others in proteins expressed at different pH and exposed to oxidative environments. The results also have implications for interpreting site-directed mutagenesis studies aimed at identifying copper binding amino acids in copper trafficking proteins.

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.

Superoxide dismutase 1 (SOD1) maturation within the cell is mainly accomplished with the SOD1-specific chaperone, CCS, a dimeric protein with three distinct domains in each monomer. We recently showed that the first domain of human CCS (hCCSD1) is responsible for copper transfer to its protein partner, human SOD1 (hSOD1). The NMR solution structure of the copper(I)-loaded form of hCCSD1 reported here contributes further to characterization of the copper-transfer mechanism to hSOD1. NMR spectroscopy was also used to examine the hSOD1 mutants C57A, C146A, and C57A/C146A, which are unable to form the structurally conserved disulfide bond in SOD1, in order to investigate the role of these cysteines during hSOD1 copper acquisition. Together, the information on both hCCS and hSOD1, along with a sequence analysis of eukaryotic CCSD1, allows us to propose important mechanistic aspects regarding the copper-transfer process from hCCS to hSOD1.

Proteases are commonly secreted by microorganisms. In some pathogens, they can play a series of functional roles during infection, including maturation of cell surface or extracellular virulence factors, interference with host cell signaling, massive host tissue destruction, and dissolution of infection-limiting clots through degradation of the host proteins devoted to the coagulation cascade. We previously reported the identification and characterization of Zmp1, a zinc-dependent metalloprotease secreted by Clostridium difficile, demonstrated that Zmp1 is able to degrade fibrinogen in vitro, and identified two residues necessary to the catalytic activity. In the present work, we solved the solution structure of Zmp1 by Nuclear Magnetic Resonance (NMR) and compared it with the recently solved X-ray structures of substrate-bound and substrate-free Zmp1, highlighting similarities and differences. We also combined the structural characterization to biochemical assays and site-directed mutagenesis, to provide new insights into the catalytic site and on the residues responsible for substrate specificity. The Zmp1 structure showed similarity to the catalytic domain of Anthrax Lethal Factor of Bacillus anthracis. Analogies and differences in the catalytic and in the substrate-binding sites of the two proteins are discussed.