Abstract
Common features of the extracellular carbohydrate-active virulence factors involved in host-pathogen interactions are their large sizes and modular complexities. This has made them recalcitrant to structural analysis, and therefore our understanding of the significance of modularity in these important proteins is lagging. Clostridium perfringens is a prevalent human pathogen that harbors a wide array of large, extracellular carbohydrate-active enzymes and is an excellent and relevant model system to approach this problem. Here we describe the complete structure of C. perfringens GH84C (NagJ), a 1001-amino acid multimodular homolog of the C. perfringens micro-toxin, which was determined using a combination of small angle x-ray scattering and x-ray crystallography. The resulting structure reveals unprecedented insight into how catalysis, carbohydrate-specific adherence, and the formation of molecular complexes with other enzymes via an ultra-tight protein-protein interaction are spatially coordinated in an enzyme involved in a host-pathogen interaction.
Highlights
Microbial and viral invaders of the human body often exploit host glycans to aid in adherence and must contend with the protective and structural sugar layers to enable invasion and further spread of the infection
The considerable number of large and multimodular carbohydrate-active enzymes produced by C. perfringens makes this organism an excellent and relevant model system for the study of complex carbohydrate-active enzymes involved in bacterial pathogenesis
The cohesin module (Coh) module is perhaps the most unique component, because it functions to recognize and bind ultratightly to dockerin modules (Doc), such as that present at the C terminus of the -toxin, and plays a role in forming higher order complexes with other large C. perfringens exo-toxins, all of which are thought to contribute to the virulence of this bacterium [13]
Summary
Materials—Unless otherwise stated, chemicals, carbohydrates, glycoproteins, and polysaccharides were purchased from Sigma. The GH84C-CBM32 modular pair structure was solved by molecular replacement by first running MOLREP [31] using the GH84C catalytic module as a search model to find the single molecule in the asymmetric unit. The structure of the Coh-FN3 modular pair was determined by molecular replacement using PHASER and the coordinates of the isolated Coh module (PDB accession 2O4E) as a search model to find the single molecule of Coh-FN3 in the asymmetric unit. The molecular replacement solution contained only ϳ50% of the asymmetric unit contents, the initial phases from restrained refinement with REFMAC were of sufficient quality for ARP/wARP to build a complete model, including the FN3 module, with docked side chains.
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