Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDFYajC

• Published in: PloS one 2017-06, Vol.12 (6), p.e0178307
• Main Authors: Hsieh, Ying-hsin, Huang, Ying-ju, Zhang, Hao, Liu, Qian, Lu, Yang, Yang, Hsiuchin, Houghton, John, Jiang, Chun, Sui, Sen-Fang, Tai, Phang C
• Format: Article
• Language: English
• Subjects: ATPases; Ion channels; Liposomes; Protein structure; Translocations (Genetics)
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0178307

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 (SecA.sub.1-493) 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 SecA.sub.619-831, which includes the "Helical Scaffold" Domain (SecA.sub.619-668) 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.