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PLoS One
2017 Jan 01;126:e0178307. doi: 10.1371/journal.pone.0178307.
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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.
Hsieh YH
,
Huang YJ
,
Zhang H
,
Liu Q
,
Lu Y
,
Yang H
,
Houghton J
,
Jiang C
,
Sui SF
,
Tai PC
.
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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.
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Fig 1. Intrinsic and lipid-stimulated ATPase activities of SecA N-terminal fragments.The assays are as described in Experimental Procedures.
Fig 2. AFM structure image of SecAs.(A) AFM images formed by SecA/SecA fragments. (B) Normal distribution of radius of ring-like structures formed by SecA/SecA N-fragments. Plots were generated by R program, adopted from http://www.r-projet.org (a free software for statistical computing and graphics), based on 100 random samples with similar means and variances of each measured datum. The efficiency of forming pore-ring structure for N640 was about 40%, for N643 or larger >70%, and for N668 >90%.
Fig 3. Function of SecA-only liposomes.(A) Channel activity. (B) Protein translocation. Assays as described in Experimental procedures.
Fig 4. Function of SecA-liposomes with SecYEG/SecDF-YajC.(A) SecA-domain-SecYEG channel activity. (B) SecA-domain with SecYEG-SecDF-YajC Channel activity. (C) Translocation activity in membranes: 1 μg SecA/fragment and 4.5 μg urea treated OmpA-depleted 773 membranes or 120 μg liposomes were used. The translocation in 100 μL were conducted under 37C for 30 min and analyzed as in Experimental procedures.
Fig 5. Complementation of SecA N-domains for AFM ring-structures.Same amounts of proteins were used: molar ratios of C34: N-fragments were about 2:1. Full length SecA was used for comparison. (A) AFM images with mean sizes and SD. (B) Size distribution as analyzed in Fig 2B. No complementation of pore structures with ovalbumin instead of C34 (data not shown).
Fig 6. Functional complementation of SecA fragments with C-terminal fragment C34.(A) Channel activity in SecA domains reconstituted with C34, SecA610-901. (B) Translocation activity in SecA domains reconstituted with C34. N609 together with C34 have no activity, and could serve as internal negative controls. The reactions were conducted with liposomes with SecYEG or SecYEG-SecDFC as indicated. 100% translocation activity is SecA with SecYEG-DFC.
Fig 7. Complementation with C-terminal domains for activities.(A) Liposomes channel activity. N643 or N668 alone has no channel activity (B) Membrane protein translocation. C34 fragment: SecA610-901. C28 fragment: SecA662-901. C30 fragment: SecA 640–901. None of SecA fragments alone has any translocation activity.
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