Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket
Authors
Liu, HuiRadhakrishnan, Padhma
Magoun, Loranne
Prabu-Jeyabalan, Moses
Campellone, Kenneth Geno
Savage, Pamela Joyce
He, Feng
Schiffer, Celia A.
Leong, John M.
UMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyDepartment of Molecular Genetics and Microbiology
Graduate School of Biomedical Sciences
Document Type
Journal ArticlePublication Date
2002-10-02Keywords
Actins; Adhesins, Bacterial; Carrier Proteins; Cell Line; Escherichia coli; Escherichia coli Proteins; Humans; Models, Molecular; *Point Mutation; Receptors, Cell Surface; Two-Hybrid System TechniquesLife Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50-residue region near the C-terminus of intimin. Alanine-scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin-Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin-Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.Source
Mol Microbiol. 2002 Sep;45(6):1557-73.
DOI
10.1046/j.1365-2958.2002.03137.xPermanent Link to this Item
http://hdl.handle.net/20.500.14038/34110PubMed ID
12354225Related Resources
ae974a485f413a2113503eed53cd6c53
10.1046/j.1365-2958.2002.03137.x