Abstract
SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the “Helical Scaffold” Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.
Highlights
Most proteins that traverse bacterial cytoplasmic membranes through the Sec-dependent pathway carry a N-terminal hydrophobic signal peptide that is cleaved during, or shortly after, translocation
We constructed a series of N-terminal fragments of SecA to determine the location of ATPase activity, lipid-stimulations and other functions
The intrinsic ATPase has been attributed to the nucleotide binding site I (NBDI), which resides within the first 220 aminoacyl residues of SecA (32, 40, see Supporting Information S1C Fig)
Summary
Most proteins that traverse bacterial cytoplasmic membranes through the Sec-dependent pathway carry a N-terminal hydrophobic signal peptide that is cleaved during, or shortly after, translocation. The prevailing model depicts the SecYEG complex as forming the protein-conducting channel with SecA acting as a peripheral ATPase [2, 6], which cycles on and off the cytoplasmic membrane, regulated by SecD-SecF [8, 9]. This model has been reviewed extensively [10,11,12,13]. The structure of SecA-SecYEG complex has been reported [14]
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