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An excess of catalytically required motions inhibits the scavenger decapping enzyme

Abstract

The scavenger decapping enzyme hydrolyzes the protective 5′ cap structure on short mRNA fragments that are generated from the exosomal degradation of mRNAs. From static crystal structures and NMR data, it is apparent that the dimeric enzyme has to undergo large structural changes to bind its substrate in a catalytically competent conformation. Here we studied the yeast enzyme and showed that the associated opening and closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by the binding of a second ligand to the enzyme, which occurs at high substrate concentrations. We designed a mutant that disrupted the allosteric pathway that links the second binding event to the dynamics and showed that this mutant enzyme is hyperactive. Our data reveal a unique mechanism of substrate inhibition in which motions that are required for catalytic activity also inhibit efficient turnover when they are present in excess.

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Figure 1: Structure of the yeast DcpS–substrate complex.
Figure 2: The scavenger decapping enzyme binds to two substrate molecules in a sequential manner.
Figure 3: Quantification of domain-flipping motions in the scavenger decapping enzyme.
Figure 4: Turnover rates of the enzyme are much slower than the flipping rates.
Figure 5: Cartoon representation of the substrate inhibition mechanism of the scavenger decapping enzyme.

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References

  1. Fersht, A. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding (W.H. Freeman, 1999).

  2. Hammes-Schiffer, S. & Benkovic, S.J. Relating protein motion to catalysis. Annu. Rev. Biochem. 75, 519–541 (2006).

    Article  CAS  Google Scholar 

  3. Boehr, D.D., Dyson, H.J. & Wright, P.E. An NMR perspective on enzyme dynamics. Chem. Rev. 106, 3055–3079 (2006).

    Article  CAS  Google Scholar 

  4. Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    Article  CAS  Google Scholar 

  5. Henzler-Wildman, K. & Kern, D. Dynamic personalities of proteins. Nature 450, 964–972 (2007).

    Article  CAS  Google Scholar 

  6. Tokuriki, N. & Tawfik, D.S. Protein dynamism and evolvability. Science 324, 203–207 (2009).

    Article  CAS  Google Scholar 

  7. McGeagh, J.D., Ranaghan, K.E. & Mulholland, A.J. Protein dynamics and enzyme catalysis: insights from simulations. Biochim. Biophys. Acta 1814, 1077–1092 (2011).

    Article  CAS  Google Scholar 

  8. van den Bedem, H. & Fraser, J.S. Integrative, dynamic structural biology at atomic resolution—it's about time. Nat. Methods 12, 307–318 (2015).

    Article  CAS  Google Scholar 

  9. Eisenmesser, E.Z., Bosco, D.A., Akke, M. & Kern, D. Enzyme dynamics during catalysis. Science 295, 1520–1523 (2002).

    Article  CAS  Google Scholar 

  10. Boehr, D.D., McElheny, D., Dyson, H.J. & Wright, P.E. The dynamic energy landscape of dihydrofolate reductase catalysis. Science 313, 1638–1642 (2006).

    Article  CAS  Google Scholar 

  11. Henzler-Wildman, K.A. et al. Intrinsic motions along an enzymatic reaction trajectory. Nature 450, 838–844 (2007).

    Article  CAS  Google Scholar 

  12. Wolf-Watz, M. et al. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat. Struct. Mol. Biol. 11, 945–949 (2004).

    Article  CAS  Google Scholar 

  13. Závodszky, P., Kardos, J., Svingor, Á. & Petsko, G.A. Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc. Natl. Acad. Sci. USA 95, 7406–7411 (1998).

    Article  Google Scholar 

  14. Agarwal, P.K., Billeter, S.R., Rajagopalan, P.T., Benkovic, S.J. & Hammes-Schiffer, S. Network of coupled promoting motions in enzyme catalysis. Proc. Natl. Acad. Sci. USA 99, 2794–2799 (2002).

    Article  CAS  Google Scholar 

  15. Röthlisberger, D. et al. Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195 (2008).

    Article  Google Scholar 

  16. Wang, Z. & Kiledjian, M. Functional link between the mammalian exosome and mRNA decapping. Cell 107, 751–762 (2001).

    Article  CAS  Google Scholar 

  17. Liu, H., Rodgers, N.D., Jiao, X. & Kiledjian, M. The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases. EMBO J. 21, 4699–4708 (2002).

    Article  CAS  Google Scholar 

  18. Malys, N. & McCarthy, J.E. Dcs2, a novel stress-induced modulator of m7GpppX pyrophosphatase activity that locates to P bodies. J. Mol. Biol. 363, 370–382 (2006).

    Article  CAS  Google Scholar 

  19. Singh, J. et al. DcpS as a therapeutic target for spinal muscular atrophy. ACS Chem. Biol. 3, 711–722 (2008).

    Article  CAS  Google Scholar 

  20. Gogliotti, R.G. et al. The DcpS inhibitor RG3039 improves survival, function and motor unit pathologies in two SMA mouse models. Hum. Mol. Genet. 22, 4084–4101 (2013).

    Article  CAS  Google Scholar 

  21. Chen, N., Walsh, M.A., Liu, Y., Parker, R. & Song, H. Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping. J. Mol. Biol. 347, 707–718 (2005).

    Article  CAS  Google Scholar 

  22. Gu, M. et al. Insights into the structure, mechanism and regulation of scavenger mRNA decapping activity. Mol. Cell 14, 67–80 (2004).

    Article  CAS  Google Scholar 

  23. Han, G.W. et al. Crystal structure of an apo mRNA decapping enzyme (DcpS) from mouse at 1.83 Å resolution. Proteins 60, 797–802 (2005).

    Article  CAS  Google Scholar 

  24. Séraphin, B. The HIT protein family: a new family of proteins present in prokaryotes, yeast and mammals. DNA Seq. 3, 177–179 (1992).

    Article  Google Scholar 

  25. Liu, S.W., Rajagopal, V., Patel, S.S. & Kiledjian, M. Mechanistic and kinetic analysis of the DcpS scavenger decapping enzyme. J. Biol. Chem. 283, 16427–16436 (2008).

    Article  CAS  Google Scholar 

  26. Pentikäinen, U., Pentikäinen, O.T. & Mulholland, A.J. Cooperative symmetric to asymmetric conformational transition of the apo form of scavenger decapping enzyme revealed by simulations. Proteins 70, 498–508 (2008).

    Article  Google Scholar 

  27. Tugarinov, V., Hwang, P.M., Ollerenshaw, J.E. & Kay, L.E. Cross-correlated relaxation enhanced 1H-13C NMR spectroscopy of methyl groups in very high-molecular-weight proteins and protein complexes. J. Am. Chem. Soc. 125, 10420–10428 (2003).

    Article  CAS  Google Scholar 

  28. Sprangers, R. & Kay, L.E. Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445, 618–622 (2007).

    Article  CAS  Google Scholar 

  29. Audin, M.J. et al. The archaeal exosome: identification and quantification of site-specific motions that correlate with cap and RNA binding. Angew. Chem. Int. Edn Engl. 52, 8312–8316 (2013).

    Article  CAS  Google Scholar 

  30. Gelis, I. et al. Structural basis for signal sequence recognition by the translocase motor SecA as determined by NMR. Cell 131, 756–769 (2007).

    Article  CAS  Google Scholar 

  31. Rosenzweig, R. & Kay, L.E. Bringing dynamic molecular machines into focus by methyl-TROSY NMR. Annu. Rev. Biochem. 83, 291–315 (2014).

    Article  CAS  Google Scholar 

  32. Wypijewska, A. et al. 7-methylguanosine diphosphate (m7GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity. Biochemistry 51, 8003–8013 (2012).

    Article  CAS  Google Scholar 

  33. Sprangers, R., Gribun, A., Hwang, P.M., Houry, W.A. & Kay, L.E. Quantitative NMR spectroscopy of supramolecular complexes: dynamic side pores in ClpP are important for product release. Proc. Natl. Acad. Sci. USA 102, 16678–16683 (2005).

    Article  CAS  Google Scholar 

  34. Amero, C. et al. A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies. J. Biomol. NMR 50, 229–236 (2011).

    Article  CAS  Google Scholar 

  35. Liu, S.W. et al. Functional analysis of mRNA scavenger decapping enzymes. RNA 10, 1412–1422 (2004).

    Article  CAS  Google Scholar 

  36. Farrow, N.A., Zhang, O., Forman-Kay, J.D. & Kay, L.E. A heteronuclear correlation experiment for simultaneous determination of 15N longitudinal decay and chemical exchange rates of systems in slow equilibrium. J. Biomol. NMR 4, 727–734 (1994).

    Article  CAS  Google Scholar 

  37. Hammes, G.G. Mechanism of enzyme catalysis. Nature 204, 342–343 (1964).

    Article  CAS  Google Scholar 

  38. Reed, M.C., Lieb, A. & Nijhout, H.F. The biological significance of substrate inhibition: a mechanism with diverse functions. Bioessays 32, 422–429 (2010).

    Article  CAS  Google Scholar 

  39. Taverniti, V. & Seraphin, B. Elimination of cap structures generated by mRNA decay involves the new scavenger mRNA decapping enzyme Aph1/FHIT together with DcpS. Nucleic Acids Res. 43, 482–492 (2015).

    Article  CAS  Google Scholar 

  40. Sinturel, F., Brechemier-Baey, D., Kiledjian, M., Condon, C. & Benard, L. Activation of 5′-3′ exoribonuclease Xrn1 by cofactor Dcs1 is essential for mitochondrial function in yeast. Proc. Natl. Acad. Sci. USA 109, 8264–8269 (2012).

    Article  CAS  Google Scholar 

  41. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  Google Scholar 

  42. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  43. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  44. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  45. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  46. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge J. Peters for excellent technical support, S. Wiesner for discussions, G. Zocher for assistance in recording diffraction data, T. Stehle for support and L. Kay for valuable comments on the manuscript. This work has received funding from the Max Planck Society and the European Research Council under the EU's Seventh Framework Programme (FP7/2007–2013), ERC grant agreement no. 616052.

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R.S. conceived the project. A.N. and R.S. designed the experiments. All authors performed experiments. R.S., A.N., U.N. and A.-L.F. analyzed and interpreted the data. R.S. wrote the manuscript and all authors commented on the manuscript.

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Correspondence to Remco Sprangers.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–7 and Supplementary Table 1. (PDF 8327 kb)

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Neu, A., Neu, U., Fuchs, AL. et al. An excess of catalytically required motions inhibits the scavenger decapping enzyme. Nat Chem Biol 11, 697–704 (2015). https://doi.org/10.1038/nchembio.1866

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