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Allosteric regulation of DegS protease subunits through a shared energy landscape

Nature Chemical Biology volume 9, pages 90–96 (2013)Cite this article

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Abstract

The PDZ domains of the trimeric DegS protease bind unassembled outer-membrane proteins (OMPs) that accumulate in the Escherichia coli periplasm. This cooperative binding reaction triggers a proteolytic cascade that activates a transcriptional stress response. To dissect the mechanism of allosteric activation, we generated hybrid DegS trimers with different numbers of PDZ domains and/or protease-domain mutations. By studying the chemical reactivity and enzymatic properties of these hybrids, we show that all subunits experience a strongly coupled energetic landscape. For example, OMP peptide binding to a single PDZ domain stimulates active site chemical modification and proteolytic cleavage in the attached and neighboring protease domains. OMP peptide binding relieves inhibitory PDZ interactions, whereas the interfaces between protease domains in the trimeric DegS core mediate positively cooperative activation driven by both substrate binding and inhibition relief.

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Figure 1: DegS and hybrid trimers containing mixtures of S and Δ subunits.
Figure 2: Basal and OMP-activated RseAP cleavage by the ΔΔΔ, SΔΔ, SSΔ and SSS enzymes.
Figure 3: OMP peptide binding activates cis and trans subunits.
Figure 4: Trimers containing Δ subunits with the Y162A mutation have reduced activity compared to otherwise isogenic trimers without this destabilizing mutation.
Figure 5: Structural determinants of stabilization of the inactive and active DegS conformations.

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  • 27 December 2012

    In the version of this article initially published online, the title contained a spelling error: "through" was misspelled as "though." The error has been corrected for the PDF and HTML versions of the article.

References

  1. Singh, N., Kuppili, R.R. & Bose, K. The structural basis of mode of activation and functional diversity: a case study with HtrA family of serine proteases. Arch. Biochem. Biophys. 516, 85–96 (2011).

    Article  CAS  Google Scholar 

  2. Grau, S. et al. Implications of the serine protease HtrA1 in amyloid precursor protein processing. Proc. Natl. Acad. Sci. USA 102, 6021–6026 (2005).

    Article  CAS  Google Scholar 

  3. Coleman, H.R., Chan, C.C., Ferris, F. & Chew, E.Y. Age-related macular degeneration. Lancet 372, 1835–1845 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  4. Milner, J.M., Patel, A. & Rowan, A.D. Emerging roles of serine proteinases in tissue turnover in arthritis. Arthritis Rheum. 58, 3644–3656 (2008).

    Article  CAS  Google Scholar 

  5. Vande Walle, L., Lamkanfi, M. & Vandenabeele, P. The mitochondrial serine protease HtrA2/Omi: an overview. Cell Death Differ. 15, 453–460 (2008).

    Article  CAS  Google Scholar 

  6. Chien, J., Campioni, M., Shridhar, V. & Baldi, A. HtrA serine proteases as potential therapeutic targets in cancer. Curr. Cancer Drug Targets 9, 451–468 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  7. Hara, K. et al. Association of HTRA1 mutations and familial ischemic cerebral small-vessel disease. N. Engl. J. Med. 360, 1729–1739 (2009).

    Article  CAS  Google Scholar 

  8. Bowden, M.A. et al. High-temperature requirement factor A3 (Htra3): a novel serine protease and its potential role in ovarian function and ovarian cancers. Mol. Cell. Endocrinol. 327, 13–18 (2010).

    Article  CAS  Google Scholar 

  9. Walsh, N.P., Alba, B.M., Bose, B., Gross, C.A. & Sauer, R.T. OMP peptide signals initiate the envelope-stress response by activating DegS protease via relief of inhibition mediated by its PDZ domain. Cell 113, 61–71 (2003).

    Article  CAS  Google Scholar 

  10. Alba, B.M. & Gross, C.A. Regulation of the Escherichia coli σE-dependent envelope stress response. Mol. Microbiol. 52, 613–619 (2004).

    Article  CAS  Google Scholar 

  11. Waller, P.R. & Sauer, R.T. Characterization of degQ and degS, Escherichia coli genes encoding homologs of the DegP protease. J. Bacteriol. 178, 1146–1153 (1996).

    Article  CAS  PubMed Central  Google Scholar 

  12. De Las Peñas, A., Connolly, L. & Gross, C.A. The σE-mediated response to extracytoplasmic stress in Escherichia coli is transduced by RseA and RseB, two negative regulators of σE. Mol. Microbiol. 24, 373–385 (1997).

    Article  Google Scholar 

  13. Missiakas, D., Mayer, M.P., Lemaire, M., Georgopoulos, C. & Raina, S. Modulation of the Escherichia coli σE (RpoE) heat-shock transcription-factor activity by the RseA, RseB and RseC proteins. Mol. Microbiol. 24, 355–371 (1997).

    Article  CAS  Google Scholar 

  14. Campbell, E.A. et al. Crystal structure of Escherichia coli σE with the cytoplasmic domain of its anti-σ RseA. Mol. Cell 11, 1067–1078 (2003).

    Article  CAS  Google Scholar 

  15. Ades, S.E., Grigorova, I.L. & Gross, C.A. Regulation of the alternative σ factor σE during initiation, adaptation, and shutoff of the extracytoplasmic heat shock response in Escherichia coli. J. Bacteriol. 185, 2512–2519 (2003).

    Article  CAS  PubMed Central  Google Scholar 

  16. Kanehara, K., Ito, K. & Akiyama, Y. YaeL (EcfE) activates the σE pathway of stress response through a site-2 cleavage of anti-σE, RseA. Genes Dev. 16, 2147–2155 (2002).

    Article  CAS  PubMed Central  Google Scholar 

  17. Akiyama, Y., Kanehara, K. & Ito, K. RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences. EMBO J. 23, 4434–4442 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  18. Li, X. et al. Cleavage of RseA by RseP requires a carboxyl-terminal hydrophobic amino acid following DegS cleavage. Proc. Natl. Acad. Sci. USA 106, 14837–14842 (2009).

    Article  CAS  Google Scholar 

  19. Flynn, J.M., Levchenko, I., Sauer, R.T. & Baker, T.A. Modulating substrate choice: the SspB adaptor delivers a regulator of the extracytoplasmic-stress response to the AAA+ protease ClpXP for degradation. Genes Dev. 18, 2292–2301 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  20. Chaba, R., Grigorova, I.L., Flynn, J.M., Baker, T.A. & Gross, C.A. Design principles of the proteolytic cascade governing the σE-mediated envelope stress response in Escherichia coli: keys to graded, buffered, and rapid signal transduction. Genes Dev. 21, 124–136 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  21. Wilken, C., Kitzing, K., Kurzbauer, R., Ehrmann, M. & Clausen, T. Crystal structure of the DegS stress sensor: how a PDZ domain recognizes misfolded protein and activates a protease. Cell 117, 483–494 (2004).

    Article  CAS  Google Scholar 

  22. Zeth, K. Structural analysis of DegS, a stress sensor of the bacterial periplasm. FEBS Lett. 569, 351–358 (2004).

    Article  CAS  Google Scholar 

  23. Sohn, J., Grant, R.A. & Sauer, R.T. Allosteric activation of DegS, a stress sensor PDZ protease. Cell 131, 572–583 (2007).

    Article  CAS  Google Scholar 

  24. Sohn, J., Grant, R.A. & Sauer, R.T. Allostery is an intrinsic property of the protease domain of DegS: implications for enzyme function and evolution. J. Biol. Chem. 285, 34039–34047 (2010).

    Article  CAS  PubMed Central  Google Scholar 

  25. Monod, J., Wyman, J. & Changeux, J.P. On the nature of allosteric transitions: a plausible model. J. Mol. Biol. 12, 88–118 (1965).

    Article  CAS  PubMed Central  Google Scholar 

  26. Sohn, J. & Sauer, R.T. OMP peptides modulate the activity of DegS protease by differential binding to active and inactive conformations. Mol. Cell 33, 64–74 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  27. Sohn, J., Grant, R.A. & Sauer, R.T. OMP peptides activate the DegS stress-sensor protease by a relief of inhibition mechanism. Structure 17, 1411–1421 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  28. Hasselblatt, H. et al. Regulation of the σE stress response by DegS: how the PDZ domain keeps the protease inactive in the resting state and allows integration of different OMP-derived stress signals upon folding stress. Genes Dev. 21, 2659–2670 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  29. Cui, Q. & Karplus, M. Allostery and cooperativity revisited. Protein Sci. 17, 1295–1307 (2008).

    Article  CAS  PubMed Central  Google Scholar 

  30. del Sol, A., Tsai, C.J., Ma, B. & Nussinov, R. The origin of allosteric functional modulation: multiple pre-existing pathways. Structure 17, 1042–1050 (2009).

    Article  CAS  PubMed Central  Google Scholar 

  31. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. USA 96, 14694–14699 (1999).

    Article  CAS  PubMed Central  Google Scholar 

  32. Kimmel, J.L. & Reinhart, G.D. Isolation of an individual allosteric interaction in tetrameric phosphofructokinase from Bacillus stearothermophilus. Biochemistry 40, 11623–11629 (2001).

    Article  CAS  Google Scholar 

  33. Schneider, F., Hammarström, P. & Kelly, J.W. Transthyretin slowly exchanges subunits under physiological conditions: a convenient chromatographic method to study subunit exchange in oligomeric proteins. Protein Sci. 10, 1606–1613 (2001).

    Article  CAS  PubMed Central  Google Scholar 

  34. Clausen, T., Kaiser, M., Huber, R. & Ehrmann, M. HTRA proteases: regulated proteolysis in protein quality control. Nat. Rev. Mol. Cell Biol. 12, 152–162 (2011).

    Article  CAS  Google Scholar 

  35. Creighton, T.E. Proteins, Structures and Molecular Properties 2nd edn. (WH Freeman & Co., New York, 1993).

  36. Eigen, M. Kinetics of reaction control and information transfer in enzymes and nucleic acids. Nobel Symp. 5, 333–369 (1967).

    Google Scholar 

  37. Hammes, G.G. & Wu, C.W. Regulation of enzyme activity. The activity of enzymes can be controlled by a multiplicity of conformational equilibria. Science 172, 1205–1211 (1971).

    Article  CAS  Google Scholar 

  38. Hagan, C.L., Silhavy, T.J. & Kahne, D. β-Barrel membrane protein assembly by the Bam complex. Annu. Rev. Biochem. 80, 189–210 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grant AI-16892 (R.T.S.). R.V.M. was supported by an NIH postdoctoral fellowship (GM097972). We thank B. Stinson, A. Olivares, J. Sohn, A. Nager, A. de Regt and S. Kim for helpful discussions.

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  1. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Randall V Mauldin & Robert T Sauer

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  1. Randall V Mauldin
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R.V.M. performed all experiments. R.V.M. and R.T.S. contributed to experimental design, interpretation and writing of the manuscript.

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Correspondence to Robert T Sauer.

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Mauldin, R., Sauer, R. Allosteric regulation of DegS protease subunits through a shared energy landscape. Nat Chem Biol 9, 90–96 (2013). https://doi.org/10.1038/nchembio.1135

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