Selective photoaffinity labeling identifies the signal peptide binding domain on SecA |
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Authors: | Musial-Siwek Monika Rusch Sharyn L Kendall Debra A |
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Affiliation: | Department of Molecular and Cell Biology, 91 North Eagleville Road, University of Connecticut, Storrs, CT 06269-3125, USA. |
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Abstract: | SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the signal peptide binding groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53 residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acid residues 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA. |
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Keywords: | HWD, helical wing domain HSD, helical scaffold domain PPXD, preprotein crosss-linking domain SSD, substrate specificity domain Bpa, ρ-benzoylphenylalanine SPBG, signal peptide binding groove WT SP, wild-type signal peptide WT(Bpa)-biotin, wild-type signal peptide with incorporated Bpa and labeled with biotin |
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