Abstract
Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase1,2,3,4, is enhanced by proton motive force5,6 and membrane-integrated SecDF7,8,9, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis4,10,11,12,13. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.
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Accession codes
Primary accessions
Biological Magnetic Resonance Data Bank
Protein Data Bank
Data deposits
The coordinates and structure factors have been deposited in the Protein Data Bank under the accession codes 3AQP for the entire TtSecDF protein and 3AQO for the P1 domain. The PDB and BMRB codes for the deposited P4 domain are 2RRN and 11426 respectively.
References
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Acknowledgements
We thank Y. Akiyama, R. Suno, Y. Morimoto, T. Minamino, K. Namba, K. Inaba, M. Hattori and H. Nishimasu for suggestions; T. Sakamoto and A. Kurabayashi for assistance with sample preparation; R. Yamasaki, M. Sano, K. Mochizuki, K. Yoshikaie, K. Imayoshi and T. Adachi for technical support; M. Homma and S. Kojima for providing the Vibrio genomic DNA; the beamline staff members at BL41XU of SPring-8 (Hyogo, Japan) and at NW12 of KEK PF-AR (Tsukuba, Japan) for technical help during data collection and M. Ibba for comments on our manuscript. This work was supported by a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to O.N., by a CREST grant from JST to K.I., by a BIRD grant from JST to H.M. and R.I., by a grant for the National Project on Protein Structural and Functional Analyses to O.N., by NIH grants to D.G.V. and by grants from MEXT to T.Tsukazaki, H.M., R.I., S.F. and K.I.
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Contributions
T.Tsukazaki performed the structural determination and the biochemical experiments with SecDF. H.M. performed the functional analyses of SecDF. Y.E. solved the crystal structure of the SecDF P1 domain and assisted with the functional analysis of SecDF. R.I., S.F., D.G.V. and O.N. assisted with the structural determination. A.D.M. performed patch clamp and pH fluorescence experiments. T.Tanaka and T.K. solved the structure of the P4 domain by NMR. A.P. and D.G.V. assisted with the crystallization and data collection of SecDF. All authors discussed the results and commented on the manuscript. O.N. and K.I. supervised the work and wrote and edited the manuscript.
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This file contains a Supplementary Discussion, Supplementary Methods, Supplementary References, Supplementary Figures 1- 9 with legends and Supplementary Tables 1-2. (PDF 1906 kb)
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Tsukazaki, T., Mori, H., Echizen, Y. et al. Structure and function of a membrane component SecDF that enhances protein export. Nature 474, 235–238 (2011). https://doi.org/10.1038/nature09980
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DOI: https://doi.org/10.1038/nature09980
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