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A redox switch shapes the Lon protease exit pore to facultatively regulate proteolysis

Nature Chemical Biology volume 11, pages 46–51 (2015)Cite this article

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Abstract

The Lon AAA+ protease degrades damaged or misfolded proteins in its intramolecular chamber. Its activity must be precisely controlled, but the mechanism by which Lon is regulated in response to different environments is not known. Facultative anaerobes in the Enterobacteriaceae family, mostly symbionts and pathogens, encounter both anaerobic and aerobic environments inside and outside the host′s body, respectively. The bacteria characteristically have two cysteine residues on the Lon protease (P) domain surface that unusually form a disulfide bond. Here we show that the cysteine residues act as a redox switch of Lon. Upon disulfide bond reduction, the exit pore of the P-domain ring narrows by ∼30%, thus interrupting product passage and decreasing activity by 80%; disulfide bonding by oxidation restores the pore size and activity. The redox switch (E°′ = −227 mV) is appropriately tuned to respond to variation between anaerobic and aerobic conditions, thus optimizing the cellular proteolysis level for each environment.

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Figure 1: Oxidized and reduced forms of the Lon P-domain ring.
Figure 2: Structural basis of the size change in the exit pore.
Figure 3: Redox control of the Lon activity.
Figure 4: Identification of the redox switch cysteine residues.
Figure 5: In vivo function of the Lon redox switch.
Figure 6

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Acknowledgements

We thank T. Nishii for assistance with the enzyme preparation and F. Amano (Osaka University of Pharmaceutical Science) for providing the E. coli AB1157 and JK405 strains. We would like to thank the beamline staff at BL26B1 and BL26B2 of SPring-8 for assistance during data collection. We wish to thank T. Nakagawa (UNISOKU, Co., Ltd.) for assistance with stopped-flow experiments. We thank A. Ishii, K. Ake, T. Imada and T. Nakayama for assistance with manuscript preparation. This work was supported in part by a Grant-in-Aid for Scientific Research (17770116) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (to W.N.). This work was also supported in part by the Targeted Proteins Research Program (TPRP) and the Platform for Drug Discovery, Informatics, and Structural Life Science, both from MEXT (to S.Y.).

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  1. yokoyama@riken.jp

Authors and Affiliations

  1. RIKEN Systems and Structural Biology Center, Yokohama, Japan

    Wataru Nishii, Mutsuko Kukimoto-Niino, Takaho Terada, Mikako Shirouzu, Tomonari Muramatsu & Shigeyuki Yokoyama

  2. RIKEN Structural Biology Laboratory, Yokohama, Japan

    Wataru Nishii, Takaho Terada, Tomonari Muramatsu & Shigeyuki Yokoyama

  3. Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama, Japan

    Mutsuko Kukimoto-Niino & Mikako Shirouzu

  4. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

    Masaki Kojima

  5. Himeji Hinomoto College, Hyogo, Japan

    Hiroshi Kihara

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  1. Wataru Nishii
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Contributions

W.N. and S.Y. designed this study, interpreted the data and wrote the manuscript. W.N. performed biochemical and physiological experiments. M.K.-N. carried out the crystallographic study. T.T. and M.S. supported crystallization. T.M. contributed to plasmid construction. M.K. and H.K. contributed to stopped-flow analysis.

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Correspondence to Wataru Nishii or Shigeyuki Yokoyama.

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Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–17 and Supplementary Tables 1–4. (PDF 11968 kb)

Supplementary Video

Redox switch of Lon. Structural changes in the exit pore of Lon between the oxidized and reduced forms are shown. (MOV 6554 kb)

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Nishii, W., Kukimoto-Niino, M., Terada, T. et al. A redox switch shapes the Lon protease exit pore to facultatively regulate proteolysis. Nat Chem Biol 11, 46–51 (2015). https://doi.org/10.1038/nchembio.1688

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