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Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines

Abstract

Hexameric ring-shaped ATPases of the AAA + (for ATPases associated with various cellular activities) superfamily power cellular processes in which macromolecular structures and complexes are dismantled or denatured, but the mechanisms used by these machine-like enzymes are poorly understood. By covalently linking active and inactive subunits of the ATPase ClpX to form hexamers, here we show that diverse geometric arrangements can support the enzymatic unfolding of protein substrates and translocation of the denatured polypeptide into the ClpP peptidase for degradation. These studies indicate that the ClpX power stroke is generated by ATP hydrolysis in a single subunit, rule out concerted and strict sequential ATP hydrolysis models, and provide evidence for a probabilistic sequence of nucleotide hydrolysis. This mechanism would allow any ClpX subunit in contact with a translocating polypeptide to hydrolyse ATP to drive substrate spooling into ClpP, and would prevent stalling if one subunit failed to bind or hydrolyse ATP. Energy-dependent machines with highly diverse quaternary architectures and molecular functions could operate by similar asymmetric mechanisms.

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Figure 1: Covalent connection of ClpX subunits to form single-chain pseudo-hexamers.
Figure 2: ClpX hexamers with 1–3 active subunits drive ClpP-mediated degradation of an unfolded substrate.
Figure 3: Protein degradation and ATPase activities of mutant enzymes.
Figure 4: Models of ATP hydrolysis.

References

  1. Gottesman, S. Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30, 465–506 (1996)

    CAS  Article  Google Scholar 

  2. Neuwald, A. F., Aravind, L., Spouge, J. L. & Koonin, E. V. AAA + : A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res. 9, 27–43 (1999)

    CAS  Google Scholar 

  3. Langer, T. AAA proteases: cellular machines for degrading membrane proteins. Trends Biochem. Sci. 25, 247–251 (2000)

    CAS  Article  Google Scholar 

  4. Ogura, T. & Wilkinson, A. J. AAA + superfamily ATPases: common structure—diverse function. Genes Cells 6, 575–597 (2001)

    CAS  Article  Google Scholar 

  5. Gottesman, S. Proteolysis in bacterial regulatory circuits. Annu. Rev. Cell Dev. Biol. 19, 565–587 (2003)

    CAS  Article  Google Scholar 

  6. Sauer, R. T. et al. Sculpting the proteome with AAA + proteases and disassembly machines. Cell 119, 9–18 (2004)

    CAS  Article  Google Scholar 

  7. Pickart, C. M. & Cohen, R. E. Proteasomes and their kin: proteases in the machine age. Nature Rev. Mol. Cell Biol. 5, 177–187 (2004)

    CAS  Article  Google Scholar 

  8. Grimaud, R., Kessel, M., Beuron, F., Steven, A. C. & Maurizi, M. R. Enzymatic and structural similarities between the Escherichia coli ATP-dependent proteases, ClpXP and ClpAP. J. Biol. Chem. 273, 12476–12481 (1998)

    CAS  Article  Google Scholar 

  9. Kim, Y. I., Burton, R. E., Burton, B. M., Sauer, R. T. & Baker, T. A. Dynamics of substrate denaturation and translocation by the ClpXP degradation machine. Mol. Cell 5, 639–648 (2000)

    CAS  Article  Google Scholar 

  10. Singh, S. K., Grimaud, R., Hoskins, J. R., Wickner, S. & Maurizi, M. R. Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP. Proc. Natl Acad. Sci. USA 97, 8898–8903 (2000)

    ADS  CAS  Article  Google Scholar 

  11. Burton, R. E., Siddiqui, S. M., Kim, Y. I., Baker, T. A. & Sauer, R. T. Effects of protein stability and structure on substrate processing by the ClpXP unfolding and degradation machine. EMBO J. 20, 3092–3100 (2001)

    CAS  Article  Google Scholar 

  12. Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl Acad. Sci. USA 98, 10584–10589 (2001)

    ADS  CAS  Article  Google Scholar 

  13. Kenniston, J. A., Baker, T. A., Fernandez, J. M. & Sauer, R. T. Linkage between ATP consumption and mechanical unfolding during the protein processing reactions of a AAA + degradation machine. Cell 114, 511–520 (2003)

    CAS  Article  Google Scholar 

  14. Bolon, D. N., Grant, R. A., Baker, T. A. & Sauer, R. T. Nucleotide-dependent substrate handoff from the SspB adaptor to the AAA + ClpXP protease. Mol. Cell 16, 343–350 (2004)

    CAS  Article  Google Scholar 

  15. Hersch, G. L., Burton, R. E., Bolon, D. N., Baker, T. A. & Sauer, R. T. Asymmetric interactions of ATP with the AAA + ClpX6 unfoldase: allosteric control of a protein machine. Cell 121, 1017–1027 (2005)

    CAS  Article  Google Scholar 

  16. Hingorani, M. M., Washington, M. T., Moore, K. C. & Patel, S. S. The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase. Proc. Natl Acad. Sci. USA 94, 5012–5017 (1997)

    ADS  CAS  Article  Google Scholar 

  17. Stitt, B. L. & Xu, Y. Sequential hydrolysis of ATP molecules bound in interacting catalytic sites of Escherichia coli transcription termination protein Rho. J. Biol. Chem. 273, 26477–26486 (1998)

    CAS  Article  Google Scholar 

  18. Stitt, B. L. Escherichia coli transcription termination factor Rho binds and hydrolyzes ATP using a single class of three sites. Biochemistry 40, 2276–2281 (2001)

    CAS  Article  Google Scholar 

  19. Bochtler, M. et al. The structures of HsIU and the ATP-dependent protease HsIU–HsIV. Nature 403, 800–805 (2000)

    ADS  CAS  Article  Google Scholar 

  20. Singleton, M. R., Sawaya, M. R., Ellenberger, T. & Wigley, D. B. Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell 101, 589–600 (2000)

    CAS  Article  Google Scholar 

  21. Zalk, R. & Shoshan-Barmatz, V. ATP-binding sites in brain p97/VCP (valosin-containing protein), a multifunctional AAA ATPase. Biochem. J. 374, 473–480 (2003)

    CAS  Article  Google Scholar 

  22. Hishida, T., Han, Y. W., Fujimoto, S., Iwasaki, H. & Shinagawa, H. Direct evidence that a conserved arginine in RuvB AAA + ATPase acts as an allosteric effector for the ATPase activity of the adjacent subunit in a hexamer. Proc. Natl Acad. Sci. USA 101, 9573–9577 (2004)

    ADS  CAS  Article  Google Scholar 

  23. Boyer, P. D. The ATP synthase—a splendid molecular machine. Annu. Rev. Biochem. 66, 717–749 (1997)

    CAS  Article  Google Scholar 

  24. Wojtyra, U. A., Thibault, G., Tuite, A. & Houry, W. A. The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function. J. Biol. Chem. 278, 48981–48990 (2003)

    CAS  Article  Google Scholar 

  25. Kim, D. Y. & Kim, K. K. Crystal structure of ClpX molecular chaperone from Helicobacter pylori. J. Biol. Chem. 278, 50664–50670 (2003)

    CAS  Article  Google Scholar 

  26. Joshi, S. A., Hersch, G. L., Baker, T. A. & Sauer, R. T. Communication between ClpX and ClpP during substrate processing and degradation. Nature Struct. Mol. Biol. 11, 404–411 (2004)

    CAS  Article  Google Scholar 

  27. Dougan, D. A., Reid, B. G., Horwich, A. L. & Bukau, B. ClpS, a substrate modulator of the ClpAP machine. Mol. Cell 9, 673–683 (2002)

    CAS  Article  Google Scholar 

  28. Levchenko, I., Seidel, M., Sauer, R. T. & Baker, T. A. A specificity-enhancing factor for the ClpXP degradation machine. Science 289, 2354–2356 (2000)

    ADS  CAS  Article  Google Scholar 

  29. Kenniston, J. A., Baker, T. A. & Sauer, R. T. Partitioning between unfolding and release of native domains during ClpXP degradation determines substrate selectivity and partial processing. Proc. Natl Acad. Sci. USA 102, 1390–1395 (2005)

    ADS  CAS  Article  Google Scholar 

  30. Lee, C., Schwartz, M. P., Prakash, S., Iwakura, M. & Matouschek, A. ATP-dependent proteases degrade their substrates by processively unraveling them from the degradation signal. Mol. Cell 7, 627–637 (2001)

    CAS  Article  Google Scholar 

  31. Gai, D., Zhao, R., Li, D., Finkielstein, C. V. & Chen, X. S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumour antigen. Cell 119, 47–60 (2004)

    CAS  Article  Google Scholar 

  32. Lee, S. Y. et al. Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA + ATPase domains. Genes Dev. 17, 2552–2563 (2003)

    CAS  Article  Google Scholar 

  33. Toth, E. A., Li, Y., Sawaya, M. R., Cheng, Y. & Ellenberger, T. The crystal structure of the bifunctional primase-helicase of bacteriophage T7. Mol. Cell 12, 1113–1123 (2003)

    CAS  Article  Google Scholar 

  34. Skordalakes, E. & Berger, J. M. Structure of Rho transcription terminator: mechanism of mRNA recognition and helicase loading. Cell 114, 135–146 (2003)

    CAS  Article  Google Scholar 

  35. Schwacha, A. & Bell, S. P. Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication. Mol. Cell 8, 1093–1104 (2001)

    CAS  Article  Google Scholar 

  36. Bowman, G. D., Goedken, E. R., Kazmirski, S. L., O'Donnell, M. & Kuriyan, J. DNA polymerase clamp loaders and DNA recognition. FEBS Lett. 579, 863–867 (2005)

    CAS  Article  Google Scholar 

  37. Sakato, M. & King, S. M. Design and regulation of the AAA + microtubule motor dynein. J. Struct. Biol. 146, 58–71 (2004)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank S. Bell, D. Bolon, R. Burton, P. Chien, C. Farrell, G. Hersch, J. Kenniston, K. McGinness, S. Moore, F. Solomon and K. Wang for help and discussions. T.A.B. is an employee of the Howard Hughes Medical Institute. This work was supported by grants from the NIH.

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Correspondence to Robert T. Sauer.

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Martin, A., Baker, T. & Sauer, R. Rebuilt AAA + motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 (2005). https://doi.org/10.1038/nature04031

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