This is a repository copy of Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/1051/ Article: WALKER, DANIEL, LANCASTER, LORNA, KLEANTHOUS, COLIN et al. (1 more author) (2004) Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3. Protein Science. pp. 1603-1611. ISSN 1469-896X https://doi.org/10.1110/PS.04658504 Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Identification of the catalytic motif of the microbial ribosome inactivating cytotoxin colicin E3 1,3 1,3 2 DANIEL WALKER, LORNA LANCASTER, RICHARD JAMES, AND COLIN KLEANTHOUS1 1Department of Biology, University of York, York YO10 5YW, UK 2Division 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 Colicins are plasmid-encoded multidomain protein antibi- to aid in competition for limited nutrient resources, but has otics that are produced by and are active against Escherichia also been implicated in the maintenance of biodiversity coli and closely related bacteria. Production of the toxin is (Kerr et al. 2002) and in bacterial adaptation (Walker et al. induced under conditions that cause damage to the bacterial 2004). Most colicins characterized to date possess either a chromosome and forms part of the bacterial SOS response pore-forming activity that causes depolarization of the cy- (Walker 1996). Accordingly, colicin production is believed toplasmic membrane or a lethal nuclease activity that spe- cifically targets rRNA, tRNA, or chromosomal DNA. The DNase-type colicins (E2, E7, E8, and E9) kill cells through Reprint requests to: Colin Kleanthous, Department of Biology, Univer- sity of York, York YO10 5YW, UK; e-mail: [email protected], fax: +44 nonspecific, magnesium-dependent cleavage of chromo- (0)1904 328825. somal DNA (Pommer et al. 2001; Walker et al. 2002). 3These authors contributed equally to this work. These enzymes belong to the HNH family of endonucleases Abbreviations: E3 rRNase, the ribonuclease domain of colicin E3; Im3, immunity protein of colicin E3; E9 DNase, the endonuclease domain of that includes the caspase-activated DNases, responsible for colicin E9; Im9, immunity protein of colicin E9; Col−, a noncytotoxic the degradation of chromatin in eukaryotic apoptosis colicin phenotype; KPi, potassium phosphate; CD, circular dichroism; (Walker et al. 2002; Scholz et al. 2003). The RNase-type ␭max, wavelength of maximum fluorescence intensity. Article published online ahead of print. Article and publication date are at colicins D and E5 cleave specific, albeit different, tRNA http://www.proteinscience.org/cgi/doi/10.1110/ps.04658504. species (Ogawa et al. 1999; Tomita et al. 2000), and colicin Protein Science (2004), 13:000–000. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society 1 Walker et al. E3 (and by homology colicins E4 and E6 and cloacin DF13) Here we extend this work to assign unambiguously active specifically cleaves at the A-site of the 16S rRNA, between site residues through extensive site-directed mutagenesis bases A1493 and G1494. This inactivates the ribosome and and in vivo cytotoxicity assays by using purified mutant arrests protein synthesis, inducing a cold shock-like tran- colicins. Mutant rRNase domains were tested in in vitro scriptional response prior to cell death (Bowman et al. 1971; ribosome inactivation assays and their structural integrity Walker et al. 2004). and stability assessed through spectroscopic methods. Our Cleavage of 16S rRNA by colicin E3 has been reported to findings support the idea that the rRNase activity of colicin occur only in the context of the intact 70S ribosome (Boon E3 is mediated in a similar way to that of barnase and other 1972), although other studies indicate that at high concen- members of the T1 ribonuclease family, with H58 and E62 trations of the toxin, both isolated 30S subunits (Ohno- of E3 rRNase acting as the acid–base pair. We identify other Iwashita and Imahori 1977) and even naked 16S rRNA residues, the mutations of which abolish or significantly (Ohno and Imahori 1978) are cleaved, although in the latter reduce cytotoxicity without significant perturbation of the case with a lack of specificity. Colicin E3 is cytotoxic only rRNase structure, giving a fuller picture of the arrangement against E. coli and closely related bacteria because more of the E3 rRNase active site. In addition, we identified a distantly related bacteria lack a suitable receptor and/or further class of mutations that led to a loss of cytotoxicity translocation system for uptake of the colicin. However, in through destabilization of the rRNase structure. Defining vitro colicin E3 is capable of inactivating ribosomes from the E3 rRNase active site has allowed us to identify a mini- distantly related bacterial species (Sidikaro and Nomura mal catalytic motif used to identify other E3-like rRNase 1973). Further to this, colicin E3 has been shown to inac- domains involved in biological processes such as plant– tivate chloroplast ribosomes from Euglena gracilis (Steege microbe interactions. et al. 1982) and has a cytostatic activity against leukemic cells (Smarda et al. 2001). Like other cytotoxic microbial Results ribonucleases, colicin E3 is of potential interest as a novel chemotherapeutic agent (for review, see Makarov and Ilin- Identification of Col− mutants skya 2003). The nuclease-type colicins are coexpressed and released Mutant colicin E3 proteins were purified in complex with in complex with a specific immunity protein that binds with the immunity protein Im3 and tested for cytotoxicity against high affinity to the cytotoxic domain and abolishes its en- colicin-sensitive E. coli cells in an agar plate spot test assay zymatic activity. The equilibrium dissociation constant (Kd) (Fig. 1). In this assay, serial fivefold dilutions of the toxin for both the colicin E3-Im3 and colicin E9-Im9 complexes are spotted onto a growing lawn of E. coli cells and cyto- has been measured as 10−14 M in 200 mM salt (Wallis et al. toxicity indicated by a clear or hazy zone in the cell lawn. 1995; Walker et al. 2003). However, the affinity of the For this in vivo activity assay, the intact colicin E3-Im3 complex formed between the isolated rRNase domain and complex was used because it displays a substantially greater Im3 is two orders of magnitude weaker (10−12 M) and this cytotoxic activity than the uncomplexed colicin (Walker et reflects additional weak interactions between Im3 and the al. 2003). We initially targeted residues identified by Carr et translocation domain of the toxin as revealed by the struc- al. (2000) as likely involved in catalysis or substrate binding ture of the colicin E3-Im3 complex (Soelaiman et al. 2001). through a structural similarity of the putative E3 rRNase Structures for the E3 rRNase domain Im3 complex and the active site with the active site of barnase. In total, 21 mutant full-length colicin E3-Im3 complex have been solved at 2.4 colicin complexes were purified and tested for cytotoxicity Å and 3.0 Å, respectively (Carr et al. 2000; Soelaiman et al. by this method. 2001). The location of the active site of colicin E3 was Spot tests showed that the mutant colicins Y52A, D55A, originally predicted through the similar structural arrange- H58A, E62A, and Y64A are devoid of cytotoxic activity ment of H58 and E62 of E3 rRNase with the catalytic resi- and the activity of the R40A, R42A, E60A, and R90A mu- dues of the nonspecific ribonuclease barnase (Carr et al.