Selective photoaffinity labeling identifies the signal peptide binding domain on SecA.
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.
Musial-Siwek
Monika
M
Department of Molecular and Cell Biology, 91 North Eagleville Road, University of Connecticut, Storrs, CT 06269-3125, USA.
Rusch
Sharyn L
SL
Kendall
Debra A
DA
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism; D050356:Lipid Metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D050505:Mutant Proteins; D019920:Photoaffinity Labels; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000069816:SEC Translocation Channels; D010649:Phenylalanine; D001710:Biotin; C053764:benzoylphenylalanine; D000469:Alkaline Phosphatase; D015951:Factor Xa; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins