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Ten catalytic snapshots of rhomboid intramembrane proteolysis from gate opening to peptide release

Nature Structural & Molecular Biology volume 26, pages 910–918 (2019)Cite this article

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Abstract

Protein cleavage inside the cell membrane triggers various pathophysiological signaling pathways, but the mechanism of catalysis is poorly understood. We solved ten structures of the Escherichia coli rhomboid protease in a bicelle membrane undergoing time-resolved steps that encompass the entire proteolytic reaction on a transmembrane substrate and an aldehyde inhibitor. Extensive gate opening accompanied substrate, but not inhibitor, binding, revealing that substrates and inhibitors take different paths to the active site. Catalysis unexpectedly commenced with, and was guided through subsequent catalytic steps by, motions of an extracellular loop, with local contributions from active site residues. We even captured the elusive tetrahedral intermediate that is uncleaved but covalently attached to the catalytic serine, about which the substrate was forced to bend dramatically. This unexpectedly stable intermediate indicates rhomboid catalysis uses an unprecedented reaction coordinate that may involve mechanically stressing the peptide bond, and could be selectively targeted by inhibitors.

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Fig. 1: Core chemical mechanism of serine protease catalysis.
Fig. 2: Comparison of precatalytic GlpG conformers.
Fig. 3: Time-resolved X-ray crystallography of aldehyde inhibitor binding and catalysis.
Fig. 4: Time-resolved X-ray crystallography of transmembrane substrate binding and cleavage.
Fig. 5: Enzyme analysis validates a catalytic role for the L5 loop in intramembrane proteolysis.
Fig. 6: Model of enzymatic actions underlying rhomboid intramembrane proteolysis.

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Data availability

Coordinates of all structures have been deposited into the Protein Data Bank under accession codes 6PJ4 (Snapshot-I1), 6PJ5 (Snapshot-I2), 6PJ7 (Snapshot-I3), 6PJ8 (Snapshot-I4), 6PJ9 (Snapshot-S1), 6PJA (Snapshot-S2), 6PJP (Snapshot-S3), 6PJQ (Snapshot-S4), 6PJR (Snapshot-S5) and 6PJU (Snapshot-S6). Source data for Fig. 5b–d are available with the paper online. Any other data are available from the authors upon reasonable request.

References

  1. Brown, M. S., Ye, J., Rawson, R. B. & Goldstein, J. L. Regulated intramembrane proteolysis: a control mechanism conserved from bacteria to humans. Cell 100, 391–398 (2000).

    Article  CAS  Google Scholar 

  2. Urban, S. SnapShot: cartography of intramembrane proteolysis. Cell 167, 1898–1898.e1 (2016).

    Article  CAS  Google Scholar 

  3. Verhelst, S. H. L. Intramembrane proteases as drug targets. FEBS J. 284, 1489–1502 (2017).

    Article  CAS  Google Scholar 

  4. Dusterhoft, S., Kunzel, U. & Freeman, M. Rhomboid proteases in human disease: mechanisms and future prospects. Biochim. Biophys. Acta 1864, 2200–2209 (2017).

    Article  CAS  Google Scholar 

  5. De Strooper, B. & Chavez Gutierrez, L. Learning by failing: ideas and concepts to tackle gamma-secretases in Alzheimer’s disease and beyond. Annu Rev. Pharmacol. Toxicol. 55, 419–437 (2015).

    Article  Google Scholar 

  6. Urban, S. Making the cut: central roles of intramembrane proteolysis in pathogenic microorganisms. Nat. Rev. Microbiol. 7, 411–423 (2009).

    Article  CAS  Google Scholar 

  7. Rawson, R. B. et al. Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol. Cell 1, 47–57 (1997).

    Article  CAS  Google Scholar 

  8. De Strooper, B. et al. A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398, 518–522 (1999).

    Article  Google Scholar 

  9. Manolaridis, I. et al. Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1. Nature 504, 301–305 (2013).

    Article  CAS  Google Scholar 

  10. Urban, S., Lee, J. R. & Freeman, M. Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107, 173–182 (2001).

    Article  CAS  Google Scholar 

  11. Kinch, L. N. & Grishin, N. V. Bioinformatics perspective on rhomboid intramembrane protease evolution and function. Biochim. Biophys. Acta 1828, 2937–2943 (2013).

    Article  CAS  Google Scholar 

  12. Stevenson, L. G. et al. Rhomboid protease AarA mediates quorum-sensing in Providencia stuartii by activating TatA of the twin-arginine translocase. Proc. Natl Acad. Sci. USA 104, 1003–1008 (2007).

    Article  CAS  Google Scholar 

  13. Baker, R. P., Wijetilaka, R. & Urban, S. Two plasmodium rhomboid proteases preferentially cleave different adhesins implicated in all invasive stages of malaria. PLoS Pathog. 2, e113 (2006).

    Article  Google Scholar 

  14. O’Donnell, R. A. et al. Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite. J. Cell Biol. 174, 1023–1033 (2006).

    Article  Google Scholar 

  15. Riestra, A. M. et al. A Trichomonas vaginalis rhomboid protease and its substrate modulate parasite attachment and cytolysis of host cells. PLoS Pathog. 11, e1005294 (2015).

    Article  Google Scholar 

  16. Lastun, V. L., Grieve, A. G. & Freeman, M. Substrates and physiological functions of secretase rhomboid proteases. Semin. Cell Dev. Biol. 60, 10–18 (2016).

    Article  CAS  Google Scholar 

  17. Spinazzi, M. & De Strooper, B. PARL: the mitochondrial rhomboid protease. Semin. Cell Dev. Biol. 60, 19–28 (2016).

    Article  CAS  Google Scholar 

  18. Hedstrom, L. Serine protease mechanism and specificity. Chem. Rev. 102, 4501–4524 (2002).

    Article  CAS  Google Scholar 

  19. Samara, N. L., Gao, Y., Wu, J. & Yang, W. Detection of reaction intermediates in Mg2+-dependent DNA synthesis and RNA degradation by time-resolved X-ray crystallography. Methods Enzymol. 592, 283–327 (2017).

    Article  CAS  Google Scholar 

  20. Radisky, E. S., Lee, J. M., Lu, C. J. & Koshland, D. E. Jr Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates. Proc. Natl Acad. Sci. USA 103, 6835–6840 (2006).

    Article  CAS  Google Scholar 

  21. Liu, B., Schofield, C. J. & Wilmouth, R. C. Structural analyses on intermediates in serine protease catalysis. J. Biol. Chem. 281, 24024–24035 (2006).

    Article  CAS  Google Scholar 

  22. Wilmouth, R. C. et al. X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate. Nat. Struct. Biol. 8, 689–694 (2001).

    Article  CAS  Google Scholar 

  23. Raper, A. T., Reed, A. J. & Suo, Z. Kinetic mechanism of DNA polymerases: contributions of conformational dynamics and a third divalent metal ion. Chem. Rev. 118, 6000–6025 (2018).

    Article  CAS  Google Scholar 

  24. Morgan, J. L. et al. Observing cellulose biosynthesis and membrane translocation in crystallo. Nature 531, 329–334 (2016).

    Article  CAS  Google Scholar 

  25. Nango, E. et al. A three-dimensional movie of structural changes in bacteriorhodopsin. Science 354, 1552–1557 (2016).

    Article  CAS  Google Scholar 

  26. Kupitz, C. et al. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser. Nature 513, 261–265 (2014).

    Article  CAS  Google Scholar 

  27. Suga, M. et al. Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543, 131–135 (2017).

    Article  CAS  Google Scholar 

  28. Sun, L., Li, X. & Shi, Y. Structural biology of intramembrane proteases: mechanistic insights from rhomboid and S2P to gamma-secretase. Curr. Opin. Struct. Biol. 37, 97–107 (2016).

    Article  CAS  Google Scholar 

  29. Strisovsky, K., Sharpe, H. J. & Freeman, M. Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates. Mol. Cell 36, 1048–1059 (2009).

    Article  CAS  Google Scholar 

  30. Urban, S. & Freeman, M. Substrate specificity of rhomboid intramembrane proteases is governed by helix-breaking residues in the substrate transmembrane domain. Mol. Cell 11, 1425–1434 (2003).

    Article  CAS  Google Scholar 

  31. Cho, S., Dickey, S. W. & Urban, S. Crystal structures and inhibition kinetics reveal a two-stage catalytic mechanism with drug design implications for rhomboid proteolysis. Mol. Cell 61, 329–340 (2016).

    Article  CAS  Google Scholar 

  32. Zoll, S. et al. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate-peptide complex structures. EMBO J. 33, 2408–2421 (2014).

    Article  CAS  Google Scholar 

  33. Dickey, S. W., Baker, R. P., Cho, S. & Urban, S. Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity. Cell 155, 1270–1281 (2013).

    Article  CAS  Google Scholar 

  34. Ticha, A. et al. Sensitive versatile fluorogenic transmembrane peptide substrates for rhomboid intramembrane proteases. J. Biol. Chem. 292, 2703–2713 (2017).

    Article  CAS  Google Scholar 

  35. Moin, S. M. & Urban, S. Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics. eLife 1, e00173 (2012).

    Article  Google Scholar 

  36. Urban, S. & Moin, S. M. A subset of membrane-altering agents and gamma-secretase modulators provoke nonsubstrate cleavage by rhomboid proteases. Cell Rep. 8, 1241–1247 (2014).

    Article  CAS  Google Scholar 

  37. Urban, S. & Wolfe, M. S. Reconstitution of intramembrane proteolysis in vitro reveals that pure rhomboid is sufficient for catalysis and specificity. Proc. Natl Acad. Sci. USA 102, 1883–1888 (2005).

    Article  CAS  Google Scholar 

  38. Zhou, Y. & Zhang, Y. Serine protease acylation proceeds with a subtle re-orientation of the histidine ring at the tetrahedral intermediate. Chem. Commun. (Camb.) 47, 1577–1579 (2011).

    Article  CAS  Google Scholar 

  39. Schechter, I. Mapping of the active site of proteases in the 1960s and rational design of inhibitors/drugs in the 1990s. Curr. Protein Pept. Sci. 6, 501–512 (2005).

    Article  CAS  Google Scholar 

  40. Baker, R. P. & Urban, S. Architectural and thermodynamic principles underlying intramembrane protease function. Nat. Chem. Biol. 8, 759–768 (2012).

    Article  CAS  Google Scholar 

  41. Baker, R. P., Young, K., Feng, L., Shi, Y. & Urban, S. Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate. Proc. Natl Acad. Sci. USA 104, 8257–8262 (2007).

    Article  CAS  Google Scholar 

  42. Xue, Y. & Ha, Y. Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate. J. Biol. Chem. 287, 3099–3107 (2012).

    Article  CAS  Google Scholar 

  43. Xue, Y. et al. Conformational change in rhomboid protease GlpG induced by inhibitor binding to its S′ subsites. Biochemistry 51, 3723–3731 (2012).

    Article  CAS  Google Scholar 

  44. Paslawski, W. et al. Cooperative folding of a polytopic alpha-helical membrane protein involves a compact N-terminal nucleus and nonnative loops. Proc. Natl Acad. Sci. USA 112, 7978–7983 (2015).

    Article  CAS  Google Scholar 

  45. Xue, Y. & Ha, Y. Large lateral movement of transmembrane helix S5 is not required for substrate access to the active site of rhomboid intramembrane protease. J. Biol. Chem. 288, 16645–16654 (2013).

    Article  CAS  Google Scholar 

  46. Baker, R. P. & Urban, S. Cytosolic extensions directly regulate a rhomboid protease by modulating substrate gating. Nature 523, 101–105 (2015).

    Article  CAS  Google Scholar 

  47. Wang, Y. & Ha, Y. Open-cap conformation of intramembrane protease GlpG. Proc. Natl Acad. Sci. USA 104, 2098–2102 (2007).

    Article  CAS  Google Scholar 

  48. Urban, S. & Baker, R. P. In vivo analysis reveals substrate-gating mutants of a rhomboid intramembrane protease display increased activity in living cells. Biol. Chem. 389, 1107–1115 (2008).

    Article  CAS  Google Scholar 

  49. Drag, M. & Salvesen, G. S. Emerging principles in protease-based drug discovery. Nat. Rev. Drug Discov. 9, 690–701 (2010).

    Article  CAS  Google Scholar 

  50. Kamp, F. et al. Intramembrane proteolysis of β-amyloid precursor protein by γ-secretase is an unusually slow process. Biophys. J. 108, 1229–1237 (2015).

    Article  CAS  Google Scholar 

  51. Szaruga, M. et al. Alzheimer’s-causing mutations shift αβ length by destabilizing gamma-secretase-abetan interactions. Cell 170, 443–456 e14 (2017).

    Article  CAS  Google Scholar 

  52. Bolduc, D. M., Montagna, D. R., Gu, Y., Selkoe, D. J. & Wolfe, M. S. Nicastrin functions to sterically hinder γ-secretase-substrate interactions driven by substrate transmembrane domain. Proc. Natl Acad. Sci. USA 113, E509–E518 (2016).

    Article  CAS  Google Scholar 

  53. Collaborative Computational Project, Number 4 The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994).

    Article  Google Scholar 

  54. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).

    Article  Google Scholar 

  55. Baker, R. P. & Urban, S. An inducible reconstitution system for the real-time kinetic analysis of protease activity and inhibition inside the membrane. Methods Enzymol. 584, 229–253 (2017).

    Article  CAS  Google Scholar 

  56. Kreutzberger, A. J. B., Ji, M., Aaron, J., Mihaljevic, L. & Urban, S. Rhomboid distorts lipids to break the viscosity-imposed speed limit of membrane diffusion. Science 363, eaao0076 (2019).

    Article  Google Scholar 

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Acknowledgements

We are grateful to current and past members of the Urban laboratory for discussions and support, as well as the expert, friendly and helpful staff at the Cornell High Energy Synchrotron Source (CHESS) for access to beam time. This work was supported by a National Institutes of Health grant (no. R01AI066025 to S.U.). All X-ray diffraction data were collected using instruments at CHESS (beamline supported by National Science Foundation grant no. DMR-0936384 and National Institutes of Health grant no. GM-103485).

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  1. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Sangwoo Cho, Rosanna P. Baker, Ming Ji & Siniša Urban

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Contributions

S.U. and S.C. conceived the research. S.U. made all DNA constructs. S.C. performed protein purification, X-ray crystallography and structure determination. R.P.B. performed protein purification, enzyme kinetics and thermostability analyses. M.J. conducted EPR spectroscopy. S.U. wrote the paper and all authors approved the final version of the manuscript.

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Correspondence to Siniša Urban.

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Peer review information Ines Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Supplementary Figure 1 Time-resolved X-ray crystallographic analysis of aldehyde inhibitor binding and catalysis.

A simplified schematic of GlpG action on a peptide aldehyde (see primary Fig. 3 for stereoimages and electron densities). Waters appear as red spheres, while the L5 loop is colored in pink throughout for emphasis. To obtain these structural snapshots, crystals of GlpG in a bicelle membrane were soaked with the Ac-VRMA-CHO peptide aldehyde for increasing lengths of time prior to freezing and X-ray diffraction analysis. In addition to the catalytic steps, the interactions that formed with the L5 loop are detailed in the lower left image (panel c), which is the same data as in panel b but includes L5 sidechain/mainchain interaction details.

Supplementary Figure 2 Time-resolved X-ray crystallographic analysis of transmembrane substrate binding and cleavage.

A simplified schematic of catalytic action by GlpG on a peptide substrate (see primary Fig. 4 for stereoimages and electron densities). To obtain these structural snapshots, crystals of GlpG in a bicelle membrane were soaked with the Ac-RKVRMAAIVFSFP-amide peptide for increasing lengths of time prior to freezing and X-ray diffraction analysis. The abrupt substrate bend is detailed in the lower left image, which is the same data as the panel above it but rotated ~90° to the right. Waters are rendered as red spheres, while disordered regions are highlighted in red (in b, e, f, h) or grey (in d) during the reaction steps.

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Cho, S., Baker, R.P., Ji, M. et al. Ten catalytic snapshots of rhomboid intramembrane proteolysis from gate opening to peptide release. Nat Struct Mol Biol 26, 910–918 (2019). https://doi.org/10.1038/s41594-019-0296-9

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