Selective Photoaffinity Labeling Identifies the Signal Peptide Binding Domain on SecA

• Published in: Journal of molecular biology 2007-01, Vol.365 (3), p.637-648
• Main Authors: Musial-Siwek, Monika, Rusch, Sharyn L., Kendall, Debra A.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Alkaline Phosphatase - chemistry; Alkaline Phosphatase - metabolism; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; Biotin - chemistry; Biotin - metabolism; Escherichia coli; Escherichia coli - metabolism; Factor Xa - chemistry; Factor Xa - metabolism; Lipid Metabolism; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - metabolism; Molecular Sequence Data; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Phenylalanine - analogs & derivatives; Phenylalanine - metabolism; photoaffinity labeling; Photoaffinity Labels - metabolism; Protein Binding; Protein Precursors - metabolism; Protein Processing, Post-Translational; Protein Sorting Signals; Protein Structure, Secondary; Protein Structure, Tertiary; protein transport; proteolysis; SEC Translocation Channels; SecA; signal peptide
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.10.027

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.