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Protein Science (2007), 16:2677-2683. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Characterization of the binding surface of the translocated intimin receptor, an essential protein for EPEC and EHEC cell adhesion

Nathan T. Ross1 and Benjamin L. Miller1,2

1 Department of Biochemistry and Biophysics and the Center for Future Health, University of Rochester, Rochester, New York 14642, USA
2 Department of Dermatology and the Center for Future Health, University of Rochester, Rochester, New York 14642, USA

(RECEIVED July 17, 2007; FINAL REVISION September 17, 2007; ACCEPTED September 24, 2007)

The translocated intimin receptor (TIR) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) is required for EPEC and EHEC infections, which cause widespread illness across the globe. TIR is translocated via a type-III secretion system into the intestinal epithelial cell membrane, where it serves as an anchor for E. coli attachment via its binding partner intimin. While many aspects of EPEC and EHEC infection are now well understood, the importance of the intermolecular contacts made between intimin and TIR have not been thoroughly investigated. Herein we report site-directed mutagenesis studies on the intimin-binding domain of EPEC TIR, and how these mutations affect TIR-intimin association, as analyzed by isothermal titration calorimetry and circular dichroism. These results show how two factors govern TIR's binding to intimin: A three-residue TIR hot spot is identified that largely mediates the interaction, and mutants that alter the beta-hairpin structure of TIR severely diminish binding affinity. In addition, peptides incorporating key TIR residues identified by mutagenesis are incapable of binding intimin. These results indicate that hot spot residues and structural orientation/preorganization are required for EPEC, and likely EHEC, TIR-intimin binding.

Keywords: EPEC; EHEC; intimin; translocated intimin receptor (TIR)


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Copyright © 2007 by The Protein Society.