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J. Biol. Chem., Vol. 278, Issue 24, 21860-21868, June 13, 2003

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Concerted Folding and Binding of a Flexible Colicin Domain to Its Periplasmic Receptor TolA^*

Gregor Anderluh   ¶, Qi Hong , Ruth Boetzel ||, Colin MacDonald ||, Geoffrey R. Moore ||, Richard Virden  and Jeremy H. Lakey  **

From the Department of Biology, Biotechnical Faculty, University of Ljubljana, Vena pot 111, 1000 Ljubljana, Slovenia, School of Biochemistry and Genetics, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, United Kingdom, ^|| School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Compared with folded structures, natively unfolded protein domains are over-represented in protein-protein and protein-DNA interactions. Such domains are common features of all colicins and are required for their translocation across the outer membrane of the target Escherichia coli cell. All of these domains bind to at least one periplasmic protein of the Tol or Ton family. Similar domains are found in Ton-dependent outer membrane transporters, indicating they may interact in a related manner. In this article we have studied binding of the colicin N translocation domain to its periplasmic receptor TolA, by fluorescence resonance energy transfer (FRET) using fluorescent probes attached to engineered cysteine residues and NMR techniques. The domain exhibits a random coil circular dichroism spectrum. However, FRET revealed that guanidinium hydrochloride denaturation caused increases in all measured intramolecular distances showing that, although natively unfolded, the domain is not extended. Furthermore NMR reported a compact hydrodynamic radius of 18 Å. Nevertheless the FRET-derived distances changed upon binding to TolA indicating a significant structural rearrangement. Using ¹H-¹⁵N NMR we show that, when bound, the peptide switches from a disordered state to an ordered state. The kinetics of binding and the associated structural change were measured by stopped-flow methods, and both events appear to occur simultaneously. The data therefore suggest that this molecular recognition involves the concerted binding and folding of a flexible but collapsed state.

Received for publication, January 14, 2003 , and in revised form, April 1, 2003.

^* This work was supported by Grants 056232, 040422, 055979, 066850, and 049735 from the Wellcome Trust and the Biotechnology and Biosciences Research Council (BBSRC). 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.

^¶ Recipient of a long-term Federation of European Biochemical Societies (FEBS) fellowship.

^** A BBSRC Research Development Fellow. To whom correspondence should be addressed. Tel.: 191-222-8865; Fax: 191-222-7424; E-mail: j.h.l{at}ncl.ac.uk.

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