Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus.

UNLABELLED: Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCE: By protecting microbes against reactive oxygen insults, SODs aid survival of many bacteria within their hosts. Despite the ubiquity and conservation of these key enzymes, notable species-specific differences relevant to pathogenesis remain undefined. To probe mechanisms that govern the functioning of Neisseria meningitidis and Brucella abortus SODs, we used X-ray structures, enzymology, modeling, and murine infection experiments. We identified virulence determinants common to the two homologs, assembly differences, and a unique metal reservoir within meningococcal SOD that stabilizes the enzyme and may provide a safeguard against copper toxicity. The insights reported here provide a rationale and a basis for SOD-specific drug design and an extension of immunogen design to target two important pathogens that continue to pose global health threats.

Impaired post-translational folding of familial ALS-linked Cu, Zn superoxide dismutase mutants.

Over 110 structurally diverse missense mutations in the superoxide dismutase (SOD1) gene have been linked to the pathogenesis of familial amyotrophic lateral sclerosis (FALS), yet the mechanism by which these lead to cytotoxicity still remains unknown. We have synthesized wild-type and mutant SOD1 in synchronized cell-free reticulocyte extracts replete with the full complement of molecular chaperones and folding facilitators that are normally required to fold this metalloenzyme. Here, we report that, despite being a small, single-domain protein, human SOD1 folds post-translationally to a hyperstable native-like conformation without a requirement for ATP-dependent molecular chaperones. SOD1 folding requires tight Zn but not Cu binding and proceeds through at least three kinetically and biochemically distinct states. We find that all 11 FALS-associated SOD1 mutants examined using this system delay the kinetics of folding, but do not necessarily preclude the formation of native-like states. These data suggest a model whereby impaired post-translational folding increases the population of on- and off-pathway folding intermediates that could provide an important source of proto-toxic protein, and suggest a unifying mechanism for SOD1-linked FALS pathogenesis.

Nickel superoxide dismutase structure and mechanism.

The 1.30 A resolution crystal structure of nickel superoxide dismutase (NiSOD) identifies a novel SOD fold, assembly, and Ni active site. NiSOD is a hexameric assembly of right-handed 4-helix bundles of up-down-up-down topology with N-terminal hooks chelating the active site Ni ions. This newly identified nine-residue Ni-hook structural motif (His-Cys-X-X-Pro-Cys-Gly-X-Tyr) provides almost all interactions critical for metal binding and catalysis, and thus will likely be diagnostic of NiSODs. Conserved lysine residues are positioned for electrostatic guidance of the superoxide anion to the narrow active site channel. Apo structures show that the Ni-hook motif is unfolded prior to metal binding. The active site Ni geometry cycles from square planar Ni(II), with thiolate (Cys2 and Cys6) and backbone nitrogen (His1 and Cys2) ligands, to square pyramidal Ni(III) with an added axial His1 side chain ligand, consistent with electron paramagentic resonance spectroscopy. Analyses of the three NiSOD structures and comparisons to the Cu,Zn and Mn/Fe SODs support specific molecular mechanisms for NiSOD maturation and catalysis, and identify important structure-function relationships conserved among SODs.

ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization.

Many point mutations in human Cu,Zn superoxide dismutase (SOD) cause familial amyotrophic lateral sclerosis (FALS), a fatal neurodegenerative disorder in heterozygotes. Here we show that these mutations cluster in protein regions influencing architectural integrity. Furthermore, crystal structures of SOD wild-type and FALS mutant H43R proteins uncover resulting local framework defects. Characterizations of beta-barrel (H43R) and dimer interface (A4V) FALS mutants reveal reduced stability and drastically increased aggregation propensity. Moreover, electron and atomic force microscopy indicate that these defects promote the formation of filamentous aggregates. The filaments resemble those seen in neurons of FALS patients and bind both Congo red and thioflavin T, suggesting the presence of amyloid-like, stacked beta-sheet interactions. These results support free-cysteine-independent aggregation of FALS mutant SOD as an integral part of FALS pathology. They furthermore provide a molecular basis for the single FALS disease phenotype resulting from mutations of diverse side-chains throughout the protein: many FALS mutations reduce structural integrity, lowering the energy barrier for fibrous aggregation.

Insights into Lou Gehrig's disease from the structure and instability of the A4V mutant of human Cu,Zn superoxide dismutase.

Mutations in human superoxide dismutase (HSOD) have been linked to the familial form of amyotrophic lateral sclerosis (FALS). Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is one of the most common neurodegenerative disorders in humans. In ALS patients, selective killing of motor neurons leads to progressive paralysis and death within one to five years of onset. The most frequent FALS mutation in HSOD, Ala4-->Val, is associated with the most rapid disease progression. Here we identify and characterize key differences in the stability between the A4V mutant protein and its thermostable parent (HSOD-AS), in which free cysteine residues were mutated to eliminate interferences from cysteine oxidation. Denaturation studies reveal that A4V unfolds at a guanidine-HCl concentration 1M lower than HSOD-AS, revealing that A4V is significantly less stable than HSOD-AS. Determination and analysis of the crystallographic structures of A4V and HSOD-AS reveal structural features likely responsible for the loss of architectural stability of A4V observed in the denaturation experiments. The combined structural and biophysical results presented here argue that architectural destabilization of the HSOD protein may underlie the toxic function of the many HSOD FALS mutations.