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Published online before print May 7, 2004, 10.1110/ps.04658504
Protein Science (2004), 13:1603-1611. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3

Daniel Walker1,3, Lorna Lancaster1,3, Richard James2 and Colin Kleanthous1

1 Department of Biology, University of York, York YO10 5YW, UK
2 Division of Microbiology and Infectious Diseases, School of Molecular Medical Sciences, University Hospital, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK

(RECEIVED January 30, 2004; FINAL REVISION March 4, 2004; ACCEPTED March 4, 2004)



Abstract

Colicin E3 is a cytotoxic ribonuclease that specifically cleaves 16S rRNA at the ribosomal A-site to abolish protein synthesis in sensitive Escherichia coli cells. We have performed extensive mutagenesis of the 96-residue colicin E3 cytotoxic domain (E3 rRNase), assayed mutant colicins for in vivo cytotoxicity, and tested the corresponding E3 rRNase domains for their ability to inactivate ribosome function in vitro. From 21 alanine mutants, we identified five positions where mutation resulted in a colicin with no measurable cytotoxicity (Y52, D55, H58, E62, and Y64) and four positions (R40, R42, E60, and R90) where mutation caused a significant reduction in cytotoxicity. Mutations that were found to have large in vivo and in vitro effects were tested for structural integrity through circular dichroism and fluorescence spectroscopy using purified rRNase domains. Our data indicate that H58 and E62 likely act as the acid–base pair during catalysis with other residues likely involved in transition state stabilization. Both the Y52 and Y64 mutants were found to be highly destabilized and this is the likely origin of the loss of their cytotoxicity. The identification of important active site residues and sequence alignments of known rRNase homologs has allowed us to identify other proteins containing the putative rRNase active site motif. Proteins that contained this active site motif included three hemagglutinin-type adhesins and we speculate that these have evolved to deliver a cytotoxic rRNase into eukaryotic cells during pathogenesis.

Keywords: colicin E3; ribonuclease; ribosome; active site; mutagenesis

Abbreviations: E3 rRNase, the ribonuclease domain of colicin E3 • Im3, immunity protein of colicin E3 • E9 DNase, the endonuclease domain of colicin E9 • Im9, immunity protein of colicin E9 • Col–, a noncytotoxic colicin phenotype • KPi, potassium phosphate • CD, circular dichroism • {lambda}max, wavelength of maximum fluorescence intensity

Reprint requests to: Colin Kleanthous, Department of Biology, University of York, York YO10 5YW, UK; e-mail: ck11{at}york.ac.uk, fax: +44 (0)1904 328825.

3 These authors contributed equally to this work.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04658504.


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