Mapping of the Signal Peptide-binding Domain of Escherichia coli SecA Using Förster Resonance Energy Transfer

Abstract

Identification of the signal peptide-binding domain within SecA ATPase is an important goal for understanding the molecular basis of SecA preprotein recognition as well as elucidating the chemo-mechanical cycle of this nanomotor during protein translocation. While recent studies have addressed this topic, the precise signal-peptide binding site on SecA remains controversial. The aim of the present study was to identify the SecA signal peptide-binding site using Forster Resonance Energy Transfer (FRET). FRET provides a more global view of the binding site and circumvents the common limitations of more genetic approaches where deletion and substitution mutagenesis can confound the correct interpretation of protein structure-function analysis. This study employs a collection of functional, monocysteine SecA mutants labeled with a donor fluorophore along with cysteine-containing, acceptor fluorophore-carrying PhoA signal peptides. Fluorescence anisotropy was utilized to determine equilibrium binding constants of 1.4 μM or 10.7 μM for the alkaline phosphatase signal peptide labeled at residue 22 or 2, respectively, for SecA, with a binding stoichiometry of one signal peptide bound per SecA protomer. Distance measurements determined for nine SecA mutants indicate that the signal peptide-binding domain encompasses a region proximal to residues 225–228, 371–375, 652–657, and 771–780 when mapped onto the recent NMR structure of SecA (Gelis, I., Bonvin, A., Keramisanou, D., Koukaki, M., Gouridis, G., Karamanou, S., Economou, A., and Kalodimos, C. (2007) Cell 131, 756–769). This places the signal peptide-binding domain within the heart of SecA, surrounded by and potentially responsive to domains important for binding nucleotide, mature portions of the preprotein, and the SecYEG channel component.Funding provided by National Institute of Health