title: Conserved SecA Signal Peptide-Binding Site Revealed by Engineered Protein Chimeras and Förster Resonance Energy Transfer.
abstract: 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.
authors: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Qi
authors: Q
authors: Zhang
authors: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Yan
authors: Y
authors: Li
authors: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Rich
authors: R
authors: Olson
authors: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Ishita
authors: I
authors: Mukerji
authors: Department of Molecular Biology and Biochemistry, Molecular Biophysics Program, Wesleyan University , Middletown, Connecticut 06459, United States.
authors: Donald
authors: D
authors: Oliver
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D002678:Chimera / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins