Characterization of the Escherichia coli SecA Signal Peptide-Binding Site

• Published in: Journal of Bacteriology 2012-01, Vol.194 (2), p.307-316
• Main Authors: Grady, Lorry M, Michtavy, Jennifer, Oliver, Donald B
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; alkaline phosphatase; Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Bacterial proteins; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bacteriology; Binding Sites; Biochemistry; Biological and medical sciences; E coli; energy transfer; Escherichia coli; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial - physiology; Gene Expression Regulation, Enzymologic; Magnetic Resonance Spectroscopy; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Microbiology; Miscellaneous; Models, Molecular; mutants; Mutation; nuclear magnetic resonance spectroscopy; Peptides; Protein Binding - physiology; Protein Conformation; Protein Sorting Signals - genetics; Protein Sorting Signals - physiology; protein transport; proteins; SEC Translocation Channels; secretion; signal peptide; Signal transduction
• ISSN: 0021-9193; 1098-5530; 1067-8832
• DOI: 10.1128/JB.06150-11

SecA signal peptide interaction is critical for initiating protein translocation in the bacterial Sec-dependent pathway. Here, we have utilized the recent nuclear magnetic resonance (NMR) and Förster resonance energy transfer studies that mapped the location of the SecA signal peptide-binding site to design and isolate signal peptide-binding-defective secA mutants. Biochemical characterization of the mutant SecA proteins showed that Ser226, Val310, Ile789, Glu806, and Phe808 are important for signal peptide binding. A genetic system utilizing alkaline phosphatase secretion driven by different signal peptides was employed to demonstrate that both the PhoA and LamB signal peptides appear to recognize a common set of residues at the SecA signal peptide-binding site. A similar system containing either SecA-dependent or signal recognition particle (SRP)-dependent signal peptides along with the prlA suppressor mutation that is defective in signal peptide proofreading activity were employed to distinguish between SecA residues that are utilized more exclusively for signal peptide recognition or those that also participate in the proofreading and translocation functions of SecA. Collectively, our data allowed us to propose a model for the location of the SecA signal peptide-binding site that is more consistent with recent structural insights into this protein translocation system.