HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 278, Issue 42, 40953-40958, October 17, 2003

This Article Full Text Full Text (PDF) All Versions of this Article: 278/42/40953    most recent M308227200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mohanty, A. K. Articles by Wiener, M. C. Search for Related Content PubMed PubMed Citation Articles by Mohanty, A. K. Articles by Wiener, M. C. Social Bookmarking                      What's this?

Enzymatic E-colicins Bind to Their Target Receptor BtuB by Presentation of a Small Binding Epitope on a Coiled-coil Scaffold^*

Arun K. Mohanty , Christopher M. Bishop , Thomas C. Bishop ¶, William C. Wimley  and Michael C. Wiener  ||

From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908-0736, the Department of Biochemistry, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, and ^¶Environmental Health Sciences, Tulane University Health Sciences Center, New Orleans, Louisiana 70112 and the Division of Pharmaceutical Sciences, Xavier University of New Orleans, New Orleans, Louisiana 70125

Toxins and viruses often initiate their attacks by binding to specific proteins on the surfaces of target cells. Bacterial toxins (e.g. bacteriocins) and viruses (bacteriophages) targeting Gram-negative bacteria typically bind to outer membrane proteins. Bacterial E-colicins target Escherichia coli by binding to the outer membrane cobalamin transporter BtuB. Colicins are tripartite molecules possessing receptor-binding, translocation, and toxin domains connected by long coiled-coil -helices. Surprisingly, the crystal structure of colicin E3 does not possess a recognizable globular fold in its receptor-binding domain. We hypothesized that the binding epitope of enzymatic E-colicins is a short loop connecting the two -helices that comprise the coiled-coil region and that this flanking coiled-coil region serves to present the loop in a binding-capable conformation. To test this hypothesis, we designed and synthesized a 34-residue peptide (E-peptide-1) corresponding to residues Ala³⁶⁶–Arg³⁹⁹ of the helix-loop-helix region of colicin E3. Cysteines placed near the ends of the peptide (I372C and A393C) enabled crosslinking for reduction of conformational entropy and formation of a peptide structure that would present the loop epitope. A fluorescent analog was also made for characterization of binding by measurement of fluorescence polarization. Our analysis shows the following. (i) E-peptide-1 is predominantly random coil in aqueous solution, but disulfide bond formation increases its -helical content in both aqueous buffer and solvents that promote helix formation. (ii) Fluorescein-labeled E-peptide-1 binds to purified BtuB in a calcium-dependent manner with a K_d of 43.6 ± 4.9 nM or 2370 ± 670 nM in the presence or absence of calcium, respectively. (iii) In the presence of calcium, cyanocobalamin (CN-Cbl) displaces E-peptide-1 with a nanomolar inhibition constant (K_i = 78.9 ± 5.6 nM). We conclude that the BtuB binding sites for cobalamins and enzymatic E-colicins are overlapping but inequivalent and that the distal loop and (possibly) the short -helical flanking regions are sufficient for high affinity binding.

Received for publication, July 28, 2003

^* This work was supported by National Institutes of Health Grants DK 59999 (to A. K. M. and M. C. W.) and GM 60000 (to W. C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia, PO Box 800736, Charlottesville, VA 22908-0736. Tel.: 434-243-2731; E-mail: mwiener{at}virginia.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				Y. Zhang, M. N. Vankemmelbeke, L. E. Holland, D. C. Walker, R. James, and C. N. Penfold Investigating Early Events in Receptor Binding and Translocation of Colicin E9 Using Synchronized Cell Killing and Proteolytic Cleavage J. Bacteriol., June 15, 2008; 190(12): 4342 - 4350. [Abstract] [Full Text] [PDF]

				E. Cascales, S. K. Buchanan, D. Duche, C. Kleanthous, R. Lloubes, K. Postle, M. Riley, S. Slatin, and D. Cavard Colicin Biology Microbiol. Mol. Biol. Rev., March 1, 2007; 71(1): 158 - 229. [Abstract] [Full Text] [PDF]

				N. G. Housden, S. R. Loftus, G. R. Moore, R. James, and C. Kleanthous Cell entry mechanism of enzymatic bacterial colicins: Porin recruitment and the thermodynamics of receptor binding PNAS, September 27, 2005; 102(39): 13849 - 13854. [Abstract] [Full Text] [PDF]

				C. N. Penfold, B. Healy, N. G. Housden, R. Boetzel, M. Vankemmelbeke, G. R. Moore, C. Kleanthous, and R. James Flexibility in the Receptor-Binding Domain of the Enzymatic Colicin E9 Is Required for Toxicity against Escherichia coli Cells J. Bacteriol., July 15, 2004; 186(14): 4520 - 4527. [Abstract] [Full Text] [PDF]