Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster

<p>Abstract</p> <p>Background</p> <p>Clostridial neurotoxins (CNTs) are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to <it>Clostridium </it>and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families.</p> <p>Results</p> <p>The <it>C. botulinum </it>strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798) located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within <it>C. botulinum </it>strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains.</p> <p>Conclusion</p> <p>Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This work provides new insights into the evolution of <it>C. botulinum </it>neurotoxins and the evolutionary mechanisms underlying the origins of virulent genes.</p

Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol

The final publication is available at Elsevier via https://doi.org/10.1016/j.ab.2019.03.007. © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.This work was supported by the Canadian Institutes of Health Research (CIHR) and National Sciences and Engineering Research Council of Canada (NSERC)

Modular Evolution and the Origins of Symmetry: Reconstruction of a Three-Fold Symmetric Globular Protein

SummaryThe high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules

The threat of instability: neurodegeneration predicted by protein destabilization and aggregation propensity.

A new method based on protein stability and aggregation propensity reveals a strong correlation between the properties of mutant Cu/Zn-superoxide dismutases associated with amyotrophic lateral sclerosis and patient survival

Misfolding and Aggregation of SOD1

<p>Wang et al.'s correlation of mutant SOD1 properties with disease duration [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060193#pbio-0060193-b006" target="_blank">6</a>] implicates two steps in the formation of aggregates of SOD1 in ALS: (1) native homodimeric metalloprotein, shown in ribbon representation (left) partially or completely unfolds to form a range of possible dimeric or monomeric aggregation-prone species, shown as a general grey irregular shape (middle), followed by (2) progressive assembly of the aggregation-prone species to form initially small soluble and later fibrillar aggregates (right panels). The structure of native SOD1 was generated using MolMol [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060193#pbio-0060193-b029" target="_blank">29</a>] and Protein Data Bank accession code 1n18. Sites of ALS-associated mutations studied by Wang et al. are shown in red; bound copper and zinc ions are shown as blue and black spheres, respectively. The right panels are transmission electron microscopy images of apo SOD1 aggregates formed in vitro that strongly resemble granular and granule-coated fibrillar SOD1 aggregates observed in ALS [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060193#pbio-0060193-b019" target="_blank">19</a>].</p