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Biochemistry. 2016 Mar 8;55(9):1291-300. doi: 10.1021/acs.biochem.5b01115. Epub 2016 Feb 19.

Conserved SecA Signal Peptide-Binding Site Revealed by Engineered Protein Chimeras and Förster Resonance Energy Transfer.

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Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , Middletown, Connecticut 06459, United States.


Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study, we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger subdomain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger subdomain of SecA, and they are also consistent with the proposed role of this subdomain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of SecYEG protein. In addition, our work highlights the utility of this system to conveniently engineer and study the interaction of SecA with any signal peptide of interest as well as its potential use for X-ray crystallographic studies given issues with exogenous signal peptide solubility.

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