3 years ago

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

Qian Liu, Yang Lu, Hsiuchin Yang, Hao Zhang, Ying-ju Huang, Phang C. Tai, Chun Jiang, Ying-hsin Hsieh, Sen-Fang Sui, John Houghton

by Ying-hsin Hsieh, Ying-ju Huang, Hao Zhang, Qian Liu, Yang Lu, Hsiuchin Yang, John Houghton, Chun Jiang, Sen-Fang Sui, Phang C. Tai

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.

Publisher URL: http://journals.plos.org/plosone/article

DOI: 10.1371/journal.pone.0178307

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