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J. Biol. Chem., Vol. 280, Issue 38, 32753-32760, September 23, 2005

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The Conformation of a Signal Peptide Bound by Escherichia coli Preprotein Translocase SecA^*

Yi-Te Chou1 and Lila M. Gierasch2

From the Chemistry and Biochemistry and Molecular Biology, University of Massachusetts, Amherst, Massachusetts 01003-04510

To understand the structural nature of signal sequence recognition by the preprotein translocase SecA, we have characterized the interactions of a signal peptide corresponding to a LamB signal sequence (modified to enhance aqueous solubility) with SecA by NMR methods. One-dimensional NMR studies showed that the signal peptide binds SecA with a moderately fast exchange rate (K_d 10^–5 M). The line-broadening effects observed from one-dimensional and two-dimensional NMR spectra indicated that the binding mode does not equally immobilize all segments of this peptide. The positively charged arginine residues of the n-region and the hydrophobic residues of the h-region had less mobility than the polar residues of the c-region in the SecA-bound state, suggesting that this peptide has both electrostatic and hydrophobic interactions with the binding pocket of SecA. Transferred nuclear Overhauser experiments revealed that the h-region and part of the c-region of the signal peptide form an -helical conformation upon binding to SecA. One side of the hydrophobic core of the helical h-region appeared to be more strongly bound in the binding pocket, whereas the extreme C terminus of the peptide was not intimately involved. These results argue that the positive charges at the n-region and the hydrophobic helical h-region are the selective features for recognition of signal sequences by SecA and that the signal peptide-binding site on SecA is not fully buried within its structure.

Received for publication, July 12, 2005

^* This work was supported by National Institutes of Health Grant GM034962 (to L. M. G.). 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.

¹ Present address: Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003. Tel.: 413-545-6094; Fax: 413-545-1289; E-mail: gierasch{at}biochem.umass.edu.

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