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Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC.
PLoS One 2017; 12(6):e0178307Plos

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.

Authors+Show Affiliations

Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.State Key Laboratory of Membrane Biology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing China.Department of Biology, Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA, United States of America.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

28575061

Citation

Hsieh, Ying-Hsin, et al. "Dissecting Structures and Functions of SecA-only Protein-conducting Channels: ATPase, Pore Structure, Ion Channel Activity, Protein Translocation, and Interaction With SecYEG/SecDF•YajC." PloS One, vol. 12, no. 6, 2017, pp. e0178307.
Hsieh YH, Huang YJ, Zhang H, et al. Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC. PLoS ONE. 2017;12(6):e0178307.
Hsieh, Y. H., Huang, Y. J., Zhang, H., Liu, Q., Lu, Y., Yang, H., ... Tai, P. C. (2017). Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC. PloS One, 12(6), pp. e0178307. doi:10.1371/journal.pone.0178307.
Hsieh YH, et al. Dissecting Structures and Functions of SecA-only Protein-conducting Channels: ATPase, Pore Structure, Ion Channel Activity, Protein Translocation, and Interaction With SecYEG/SecDF•YajC. PLoS ONE. 2017;12(6):e0178307. PubMed PMID: 28575061.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC. AU - Hsieh,Ying-Hsin, AU - Huang,Ying-Ju, AU - Zhang,Hao, AU - Liu,Qian, AU - Lu,Yang, AU - Yang,Hsiuchin, AU - Houghton,John, AU - Jiang,Chun, AU - Sui,Sen-Fang, AU - Tai,Phang C, Y1 - 2017/06/02/ PY - 2017/03/03/received PY - 2017/05/10/accepted PY - 2017/6/3/entrez PY - 2017/6/3/pubmed PY - 2017/9/16/medline SP - e0178307 EP - e0178307 JF - PloS one JO - PLoS ONE VL - 12 IS - 6 N2 - 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. SN - 1932-6203 UR - https://www.unboundmedicine.com/medline/citation/28575061/Dissecting_structures_and_functions_of_SecA_only_protein_conducting_channels:_ATPase_pore_structure_ion_channel_activity_protein_translocation_and_interaction_with_SecYEG/SecDF•YajC_ L2 - http://dx.plos.org/10.1371/journal.pone.0178307 DB - PRIME DP - Unbound Medicine ER -