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J. Biol. Chem., Vol. 281, Issue 45, 34072-34085, November 10, 2006

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Cysteine-scanning Mutagenesis and Disulfide Mapping Studies of the Conserved Domain of the Twin-arginine Translocase TatB Component^*

Philip A. Lee¹, George L. Orriss², Grant Buchanan², Nicholas P. Greene², Peter J. Bond, Claire Punginelli, Rachael L. Jack^¶, Mark S. P. Sansom, Ben C. Berks³, and Tracy Palmer^¶4

From the Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, and ^¶School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

The cytoplasmic membrane protein TatB is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. Together with the TatC component it forms a complex that functions as a membrane receptor for substrate proteins. Structural predictions suggest that TatB is anchored to the membrane via an N-terminal transmembrane -helix that precedes an amphipathic -helical section of the protein. From truncation analysis it is known that both these regions of the protein are essential for function. Here we construct 31 unique cysteine substitutions in the first 42 residues of TatB. Each of the substitutions results in a TatB protein that is competent to support Tat-dependent protein translocation. Oxidant-induced disulfide cross-linking shows that both the N-terminal and amphipathic helices form contacts with at least one other TatB protomer. For the transmembrane helix these contacts are localized to one face of the helix. Molecular modeling and molecular dynamics simulations provide insight into the possible structural basis of the transmembrane helix interactions. Using variants with double cysteine substitutions in the transmembrane helix, we were able to detect cross-links between up to five TatB molecules. Protein purification showed that species containing at least four cross-linked TatB molecules are found in correctly assembled TatBC complexes. Our results suggest that the transmembrane helices of TatB protomers are in the center rather than the periphery of the TatBC complex.

Received for publication, August 1, 2006

^* This work was supported by Biotechnology and Biological Sciences Research Council Grants B14749 [GenBank] and 43/P16795, and a studentship (to P. A. L.), by the Medical Research Council via a studentship (to N. P. G.), and a senior nonclinical fellowship award (to T. P.). Research in the Samson Laboratory was funded by the Biotechnology and Biological Sciences Research Council and the Wellcome Trust. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods and supplemental Refs. 1–20.

¹ Present address: Dept. of Chemical Engineering, University of Texas, Austin, TX 78712.

² These authors contributed equally to this work.

³ To whom correspondence may be addressed. Tel.: 44-1865-275250; Fax: 44-1865-275259; E-mail: ben.berks{at}bioch.ox.ac.uk. ⁴ To whom correspondence may be addressed: Tel.: 44-1603-450726, Fax: 44-1603-450778; E-mail: tracy.palmer{at}bbsrc.ac.uk.

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