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Structural analysis of an enterohemorrhagic Escherichia coli metalloprotease effector Yu, Angel Chia-yu


Mucins are proteins that contain dense clusters of α-O-GalNAc-linked carbohydrate chains and are the major component of the mucosal barrier that lines the mammalian gastrointestinal tract from mouth to gut. A critical biological function of mucins is to protect the underlying epithelial cells from infection. Enterohemorrhagic Escherichia. coli O157:H7 (EHEC), a bacterial pathogen that causes severe food and water borne disease, is capable of breaching this barrier and adhering to intestinal epithelial cells during infection. StcE (secreted protease of C1-esterase inhibitor) is a ~100 kDa zinc metalloprotease virulence factor secreted by EHEC and plays a pivotal role in remodelling the mucosal lining during EHEC pathogenesis. StcE also dampens the host immune response by targeting the mucinlike region of C1-INH, a key complement regulator of innate immunity. To obtain further mechanistic insight into StcE function, I have determined the crystal structure of the fulllength protease to 2.5Å resolution. This structure shows that StcE adopts a dynamic, multidomain architecture featuring an unusually large substrate binding cleft. Electrostatic surface analysis reveals a prominent polarized charge distribution highly suggestive of an electrostatic role in substrate targeting. The observation of key conserved motifs in the active site allows us to propose the structural basis for the specific recognition of α-O-glycan containing substrates, which have been confirmed by glycan array screening to be Oglycosylation of the mucin-type. Complementary biochemical analysis employing domain variants of StcE further extends our understanding of the substrate binding stoichiometry and distinct substrate specificity of this important virulence-associated metalloprotease.

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