The human fungal pathogens Candida albicans and Histoplasma capsulatum have been reported to protect against the oxidative burst of host innate immune cells using a family of extracellular proteins with similarity to Cu/Zn superoxide dismutase 1 (SOD1). We report here that these molecules are widespread throughout fungi and deviate from canonical SOD1 at the primary, tertiary, and quaternary levels. The structure of C. albicans SOD5 reveals that although the β-barrel of Cu/Zn SODs is largely preserved, SOD5 is a monomeric copper protein that lacks a zinc-binding site and is missing the electrostatic loop element proposed to promote catalysis through superoxide guidance. Without an electrostatic loop, the copper site of SOD5 is not recessed and is readily accessible to bulk solvent. Despite these structural deviations, SOD5 has the capacity to disproportionate superoxide with kinetics that approach diffusion limits, similar to those of canonical SOD1. In cultures of C. albicans, SOD5 is secreted in a disulfide-oxidized form and apo-pools of secreted SOD5 can readily capture extracellular copper for rapid induction of enzyme activity. We suggest that the unusual attributes of SOD5-like fungal proteins, including the absence of zinc and an open active site that readily captures extracellular copper, make these SODs well suited to meet challenges in zinc and copper availability at the host-pathogen interface. E ukaryotes are known to express two highly related classes of copper-containing superoxide dismutase (SOD) enzymes that play widespread roles in oxidative stress resistance and signaling. These two classes include an intracellular, largely cytosolic SOD1 (1) and an extracellular SOD (EC-SOD) (2), both of which are bimetallic enzymes with copper and zinc cofactors. The redox active copper catalyzes the disproportionation of superoxide anion to oxygen and hydrogen peroxide, whereas the zinc helps stabilize the protein (3-5) and promotes pH independence of the reaction (6-8). Additionally, all eukaryotic copper and zinc SODs contain an active site channel that consists of a cluster of charges in loop VII that culminate with an invariant arginine adjacent to the copper site. This element, commonly known as the electrostatic loop, is proposed to provide long-and short-range guidance for the superoxide substrate, thereby facilitating the remarkable kinetics of the SOD reaction (2, 9, 10). SOD1 is among the fastest enzymes known, with rates (10 9 M −1 s −1 ) that approach diffusion limits (9, 11, 12). Very recently, certain pathogenic fungi have been reported to express a class of extracellular proteins that are homologous to SOD1 and are covalently attached to the cell wall through GPI anchors. These SODs were first described for the opportunistic fungal pathogen Candida albicans. C. albicans is a common commensal microbe of the human gut, but under conditions of a weakened immune system, the organism can become invasive and pathogenic, with infections ranging from mild mucosal candidiasis to life-threatening system...

ALS is a terminal disease of motor neurons that is characterized by accumulation of proteinaceous deposits in affected cells. Pathological deposition of mutated Cu/Zn superoxide dismutase (SOD1) accounts for ∼20% of the familial ALS (fALS) cases. However, understanding the molecular link between mutation and disease has been difficult, given that more than 140 different SOD1 mutants have been observed in fALS patients. In addition, the molecular origin of sporadic ALS (sALS) is unclear. By dissecting the amino acid sequence of SOD1, we identified four short segments with a high propensity for amyloid fibril formation. We find that fALS mutations in these segments do not reduce their propensity to form fibrils. The atomic structures of two fibril-forming segments from the C terminus, 101 impaired nucleation and fibril growth of full-length protein, confirming that these segments participate in aggregate formation. Our hypothesis is that improper protein maturation and incompletely folded states that render these aggregation-prone segments available for interaction offer a common molecular pathway for sALS and fALS.protein aggregation | peptide structure | amyotrophic lateral sclerosis A LS is a progressive neurodegenerative disease that affects motor neurons, often causing death within 2 to 5 years. Ninety percent of ALS cases are sporadic (sALS), and their cause is unknown (1). However, the remaining 10% of ALS cases are inherited familial ALS (fALS), ∼20% of which are linked to mutations in the Cu/Zn superoxide dismutase (SOD1) gene.Mature SOD1 is a 32-kDa homodimeric metalloenzyme, in which each monomer contains a copper ion, zinc ion, and one intrasubunit disulfide bond (2) (Fig. 1A). SOD1 is one of the most abundant proteins in cells, serving to protect organisms against oxidative damage. The loss of protein function does not necessarily lead to disease because SOD1-deficient mice develop mild impairments that are not observed in ALS (3). Instead, the mutated SOD1 seems to have a toxic gain of function that leads to the pathologies of disease. To date, more than 140 dominant disease-related mutations that span nearly the whole protein sequence have been described (http://alsod.iop.kcl.ac.uk/Als/).Several studies suggest sALS and fALS have common mechanisms of pathogenesis associated with accumulation of misfolded SOD1 (4). Evidence to support this has shown that insoluble protein aggregates found in both fALS (5) and sALS (6) patients were SOD1 immunoreactive. Scientists have created transgenic mice that express human SOD1 mutations found in fALS. The mice exhibit behavioral and cellular symptoms similar to human ALS (7), including accumulation of insoluble aggregates (8). In addition, expression in mice of heterozygous wild-type/ mutant SOD1 augments disease symptoms relative to homozygous mutant animals (7), implying that the wild-type protein produced by the allele carrying the normal gene enhances the toxicity of the mutant protein in fALS. It was also shown that transgenic mice overexpressing wild-typ...

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 threedimensional 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.

Evidence of misfolded wild-type superoxide dismutase 1 (SOD1) has been detected in spinal cords of sporadic ALS (sALS) patients, suggesting an etiological relationship to SOD1-associated familial ALS (fALS). Given that there are currently a number of promising therapies under development that target SOD1, it is of critical importance to better understand the role of misfolded SOD1 in sALS. We previously demonstrated the permissiveness of the G85R-SOD1:YFP mouse model for MND induction following injection with tissue homogenates from paralyzed transgenic mice expressing SOD1 mutations. This prompted us to examine whether WT SOD1 can self-propagate misfolding of the G85R-SOD1:YFP protein akin to what has been observed with mutant SOD1. Using the G85R-SOD1:YFP mice, we demonstrate that misfolded conformers of recombinant WT SOD1, produced in vitro, induce MND with a distinct inclusion pathology. Furthermore, the distinct pathology remains upon successive passages in the G85R-SOD1:YFP mice, strongly supporting the notion for conformation-dependent templated propagation and SOD1 strains. To determine the presence of a similar misfolded WT SOD1 conformer in sALS tissue, we screened homogenates from patients diagnosed with sALS, fALS, and non-ALS disease in an organotypic spinal cord slice culture assay. Slice cultures from G85R-SOD1:YFP mice exposed to spinal homogenates from patients diagnosed with ALS caused by the A4V mutation in SOD1 developed robust inclusion pathology, whereas spinal homogenates from more than 30 sALS cases and various controls failed. These findings suggest that mutant SOD1 has prion-like attributes that do not extend to SOD1 in sALS tissues.

Human antibody recognition of Chlamydia trachomatis plasmid-encoded Pgp3 protein is dependent on the native conformation of Pgp3. The structural basis for the conformation dependence and the function of Pgp3 remain unknown. Here, we report that Pgp3 trimerization is required for the recognition of Pgp3 by human antibodies. In a native polyacrylamide gel, Pgp3 purified from a bacterial expression system migrated as stable trimers that were dissociated into monomers only by treatment with urea or sodium dodecyl sulfate (SDS) but not nonionic detergents. Human antibodies recognized trimeric but not monomeric Pgp3, suggesting that Pgp3 is presented to the human immune system as trimers during C. trachomatis infection. The endogenous Pgp3 secreted into the chlamydial outer membrane complex or host cell cytosol is always trimerized. Intact Pgp3 trimers were eluted from the outer membrane complex by a combination of nonionic detergents with reducing agents but not by the presence of either alone. These observations have provided important information for further understanding the role of Pgp3 in chlamydial pathogenesis and potentially optimizing Pgp3 as a subunit vaccine candidate antigen.

In eukaryotes the bimetallic Cu/Zn superoxide dismutase (SOD) enzymes play important roles in the biology of reactive oxygen species by disproportionating superoxide anion. Recently, we reported that the fungal pathogen Candida albicans expresses a novel copper-only SOD, known as SOD5, that lacks the zinc cofactor and electrostatic loop (ESL) domain of Cu/Zn-SODs for substrate guidance. Despite these abnormalities, C. albicans SOD5 can disproportionate superoxide at rates limited only by diffusion. Here we demonstrate that this curious copper-only SOD occurs throughout the fungal kingdom as well as in phylogenetically distant oomycetes or "pseudofungi" species. It is the only form of extracellular SOD in fungi and oomycetes, in stark contrast to the extracellular Cu/Zn-SODs of plants and animals. Through structural biology and biochemical approaches we demonstrate that these copper-only SODs have evolved with a specialized active site consisting of two highly conserved residues equivalent to SOD5 Glu-110 and Asp-113. The equivalent positions are zinc binding ligands in Cu/Zn-SODs and have evolved in copper-only SODs to control catalysis and copper binding in lieu of zinc and the ESL. Similar to the zinc ion in Cu/Zn-SODs, SOD5 Glu-110 helps orient a key copper-coordinating histidine and extends the pH range of enzyme catalysis. SOD5 Asp-113 connects to the active site in a manner similar to that of the ESL in Cu/Zn-SODs and assists in copper cofactor binding. Copper-only SODs are virulence factors for certain fungal pathogens; thus this unique active site may be a target for future anti-fungal strategies.

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases. mycoplasma cytotoxin | single-crystal X-ray diffraction | ADP-ribosyltransferase | vacuolation | reactive airway disease

Background: Pgp3 is an immunogenic protein secreted by Chlamydia trachomatis. Results: The trimeric Pgp3 structure reveals globular domains connected by a triple helical coiled-coil. Conclusion: The C-terminal domains resemble tumor necrosis factor, the helical coiled-coil has an unusual twist, and the N-terminal domain is a fusion of virus-like structural motifs. Significance: The Pgp3 structure provides insight into its role in chlamydial pathogenesis.