Структура бычьего шаперонина TRiC/CCT в открытой конформации по данным криоэлектронной микроскопии

В этой работе были подобраны условия для получения образца эукариотического шаперонина TRiC, пригодного для изучения методом криоэлектронной микроскопии. С помощью метода дифференциальной сканирующей (время-разрешенной) флуориметрии была охарактеризована температурная стабильность образцов белка при разных концентрациях соли и глицерина, а затем выбранные условия использовались для подготовки образца для микроскопии. В результате съемки и обработки изображений получена структура бычьего TRiC в открытой конформации с разрешением 4,42 Å.

1. Gestaut D., Limatola A., Joachimiak L., Frydman J. The ATP-powered gymnastics of TRiC/CCT: an asymmetric protein folding machine with a symmetric origin story. Curr. Opin. Struct. Biol. 2019;55:50–58.

2. Yam A.Y., Xia Y., Lin H.T.J., Burlingame A., Gerstein M., Program B. Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly-made proteins with complex topologies. Nat. Struct. Mol. Biol. 2008;15(12):1255–1262.

3. Bigotti M.G., Clarke A.R. Chaperonins: The hunt for the Group II mechanism. Arch. Biochem. Biophys. 2008;474(2):331–339.

4. Spiess C., Meyer A.S., Reissmann S., Frydman J. Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol. 2004;14(11):598–604.

5. Skjærven L., Cuellar J., Martinez A., Valpuesta J.M. Dynamics, flexibility, and allostery in molecular chaperonins. FEBS Lett. 2015;589(19):2522–2532.

6. Reissmann S., Joachimiak L.A., Chen B., Meyer A.S., Nguyen A., Frydman J. A Gradient of ATP affinities generates an asymmetric power stroke driving the chaperonin TRIC/CCT folding cycle. Cell Rep. 2012;2(4):866–877.

7. Reissmann S., Parnot C., Booth C.R., Chiu W., Frydman J. Essential function of the built-in lid in the allosteric regulation of eukaryotic and archaeal chaperonins. Nat. Struct. Mol. Biol. 2007;14(5):432–440.

8. Meyer A.S., Gillespie J.R., Walther D., Millet I.S., Doniach S., Frydman J. Closing the folding chamber of the eukaryotic chaperonin requires the transition state of ATP hydrolysis. Cell. 2003;113(3):369–381.

9. Cong Y., Schröder G.F., Meyer A.S., Jakana J., Ma B., Dougherty M.T., Schmid M.F., Reissmann S., Levitt M., Ludtke S.L., Frydman J., Chiu W. Symmetry-free cryo-EM structures of the chaperonin TRiC along its ATPase-driven conformational cycle. EMBO J. 2012;31(3):720–730.

10. Joachimiak L.A., Walzthoeni T., Liu C.W., Aebersold R., Frydman J. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT. Cell. 2014;159(5):1042–1055.

11. Balch W.E., Morimoto R.I., Dillin A., Kelly J.W. Adapting proteostasis for disease intervention. Science. 2008;319(5865):916–919.

12. Bouhouche A., Benomar A., Bouslam N., Chkili T., Yahyaoui M. Mutation in the epsilon subunit of the cytosolic chaperonin-containing t-complex peptide-1 (Cct5) gene causes autosomal recessive mutilating sensory neuropathy with spastic paraplegia. J. Med. Genet. 2006;43(5):441–443.

13. Kasembeli M., Lau W.C.Y., Roh S.H., Eckols T.K., Frydman J., Chiu W., Tweardy D.J. Modulation of STAT3 folding and function by TRiC/CCT chaperonin. PLoS Biol. 2014;12(4):e1001844.

14. Trinidad A.G., Muller P.A.J., Cuellar J., Klejnot M., Nobis M., Valpuesta J.M., Vousden K.H. Interaction of p53 with the CCT complex promotes protein folding and wild-type p53 activity. Mol. Cell. 2013;50(6):805–817.

15. Feldman D.E., Thulasiraman V., Ferreyra R.G., Frydman J. Formation of the VHL-elongin BC tumor suppressor complex is mediated by the chaperonin TRiC. Mol. Cell. 1999;4(6):1051–1061.

16. Feldman D.E., Spiess C., Howard D.E., Frydman J. Tumorigenic Mutations in VHL disrupt folding in vivo by interfering with chaperonin binding. Mol. Cell. 2003;12(5):1213–1224.

17. Leitner A., Joachimiak L.A., Bracher A., Mönkemeyer L., Walzthoeni T., Chen B., Pechmann S., Holmes S., Cong Y., Ma B., Ludtke S., Chiu W., Hartl F.U., Aebersold R., Frydman J. The molecular architecture of the eukaryotic chaperonin TRiC/CCT. Struct. Lond. Engl. 1993. 2012;20(5):814–825.

18. Brandvold K.R., Morimoto R.I. The chemical biology of molecular chaperones – implications for modulation of proteostasis. J. Mol. Biol. 2015;427(18):2931–2947.

19. Tam S., Spiess C., Auyeung W., Joachimiak L., Chen B., Poirier M.A., Frydman J. The chaperonin TRiC blocks a huntingtin sequence element that promotes the conformational switch to aggregation. Nat. Struct. Mol. Biol. 2009;16(12):1279–1285.

20. Tam S., Geller R., Spiess C., Frydman J. The chaperonin TRiC controls polyglutamine aggregation and toxicity through subunit-specific interactions. Nat. Cell Biol. 2006;8(10):1155–1162.

21. Kitamura A., Kubota H., Pack C.G., Matsumoto G., Hirayama S., Takahashi Y., Kimura H., Kinjo M., Morimoto R.I., Nagata K. Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state. Nat. Cell Biol. 2006;8(10):1163–1170.

22. Behrends C., Langer C.A., Boteva R., Böttcher U.M., Stemp M.J., Schaffar G., Rao B.V., Giese A., Kretzschmar H., Siegers K., Hartl F.U. Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers. Mol. Cell. 2006;23(6):887–897.

23. Sot B., Rubio-Muñoz A., Leal-Quintero A., Martínez-Sabando J., Marcilla M., Roodveldt C., Valpuesta J.M. The chaperonin CCT inhibits assembly of α-synuclein amyloid fibrils by a specific, conformationdependent interaction. Sci. Rep. 2017;7(1):40859.

24. Yébenes H., Mesa P., Muñoz I.G., Montoya G., Valpuesta J.M. Chaperonins: two rings for folding. Trends Biochem. Sci. 2011;36(8):424–432.

25. Jin M., Han W., Liu C., Zang Y., Li J., Wang F., Wang Y., Cong Y. An ensemble of cryo-EM structures of TRiC reveal its conformational landscape and subunit specificity. Proc. Natl. Acad. Sci. U.S.A. 2019;116(39):19513–19522.

26. Cong Y., Baker M.L., Jakana J., Woolford D., Miller E.J., Reissmann S., Kumar R.N., Redding-Johanson A.M., Batth T.S., Mukhopadhyay A., Ludtke S.J., Frydman J., Chiu W. 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proc. Natl. Acad. Sci. U.S.A. 2010;107(11):4967–4972.

27. Liu C., Jin M., Wang S., Han W., Zhao Q., Wang Y., Xu C., Diao L., Yin Y., Peng C., Bao L., Wang Y., Cong Y. Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM. Commun. Biol. 2023;6(1):531.

28. Leiske D.L., Shieh I.C., Tse M.L. A method to measure protein unfolding at an air–liquid interface. Langmuir. 2016;32(39):9930–9937.

29. Daban J.R. Fluorescent labeling of proteins with Nile red and 2-methoxy-2,4-diphenyl-3(2H)-furanone: Physicochemical basis and application to the rapid staining of sodium dodecyl sulfate polyacrylamide gels and Western blots. Electrophoresis. 2001;22(5):874–880.

30. Kudryavtseva S.S., Stroylova Y.Y., Kurochkina L.P., Muronetz V.I. The chaperonin TRiC is blocked by native and glycated prion protein. Arch. Biochem. Biophys. 2020;683:108319.

31. Tegunov D., Cramer P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat. Methods. 2019;16(11):1146–1152.

32. Punjanji A., Rubinstein J.L., Fleet D.A., Brubaker M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods. 2017;14(3):290–296.

33. Zivanov J., Nakane T., Forsberg B.B.O., Kimanius D., Hagen W.J.J.H., Lindahl E., Scheres S.H.W.H. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife. 2018;7:e42166.

34. Grant T., Rohou A., Grigorieff N. CisTEM, userfriendly software for single-particle image processing. eLife. 2018;7:e35383.

35. Croll T.I. ISOLDE: A physically realistic environment for model building into low-resolution electron-density maps. Acta Crystallogr. Sect. Struct. Biol. 2018;74(6):519–530.

36. Afonine P.V., Poon B.K., Read R.J., Sobolev O.V., Terwilliger T.C., Urzhumtsev A., Adams P.D. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. Sect. Struct. Biol. 2018;74(6):531–544.

37. Zang Y., Jin M., Wang H., Cui Z., Kong L., Liu C., Cong Y. Staggered ATP binding mechanism of eukaryotic chaperonin TRiC (CCT) revealed through high-resolution cryo-EM. Nat. Struct. Mol. Biol. 2016;23(12):1083–1091.

38. Muñoz I.G., Yébenes H., Zhou M., Mesa P., Serna M., Park A.Y., Bragado-Nilsson E., Beloso A., De Cárcer G., Malumbres M., Robinson C.V., Valpuesta J.M., Montoya G. Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin. Nat. Struct. Mol. Biol. 2011;18(1):14–20.