Human CCT4 and CCT5 Chaperonin Subunits Expressed in Escherichia Coli Form Biologically Active Homo-oligomers

Overview

Journal J Biol Chem

Specialty Biochemistry

Date 2013 Apr 25

PMID 23612981

Citations 41

Authors

Oksana A Sergeeva

Bo Chen

Cameron Haase-Pettingell

Steven J Ludtke

Wah Chiu

Jonathan A King

Affiliations

Soon will be listed here.

Abstract

Chaperonins are a family of chaperones that encapsulate their substrates and assist their folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP-1 ring complex (TRiC), is a hetero-oligomeric complex composed of two rings, each formed from eight different CCT (chaperonin containing TCP-1) subunits. Each CCT subunit may have distinct substrate recognition and ATP hydrolysis properties. We have expressed each human CCT subunit individually in Escherichia coli to investigate whether they form chaperonin-like double ring complexes. CCT4 and CCT5, but not the other six CCT subunits, formed high molecular weight complexes within the E. coli cells that sedimented about 20S in sucrose gradients. When CCT4 and CCT5 were purified, they were both organized as two back-to-back rings of eight subunits each, as seen by negative stain and cryo-electron microscopy. This morphology is consistent with that of the hetero-oligomeric double-ring TRiC purified from bovine testes and HeLa cells. Both CCT4 and CCT5 homo-oligomers hydrolyzed ATP at a rate similar to human TRiC and were active as assayed by luciferase refolding and human γD-crystallin aggregation suppression and refolding. Thus, both CCT4 and CCT5 homo-oligomers have the property of forming 8-fold double rings absent the other subunits, and these complexes carry out chaperonin reactions without other partner subunits.

Citing Articles

Molecular, structural, and functional characterization of delta subunit of T-complex protein-1 from .

Anand A, Gautam G, Srivastava G, Yadav S, Ramalingam K, Siddiqi M Infect Immun. 2024; 92(10):e0023424.

PMID: 39248465 PMC: 11475657. DOI: 10.1128/iai.00234-24.

Biomarker screening using integrated bioinformatics for the development of "normal-impaired glucose intolerance-type 2 diabetes mellitus".

Luo D, Gao X, Zhu X, Xu J, Gao P, Zou J Sci Rep. 2024; 14(1):4558.

PMID: 38402348 PMC: 10894242. DOI: 10.1038/s41598-024-55199-y.

A hierarchical assembly pathway directs the unique subunit arrangement of TRiC/CCT.

Betancourt Moreira K, Collier M, Leitner A, Li K, Lachapel I, McCarthy F Mol Cell. 2023; 83(17):3123-3139.e8.

PMID: 37625406 PMC: 11209756. DOI: 10.1016/j.molcel.2023.07.031.

Histopathology of Skeletal Muscle in a Distal Motor Neuropathy Associated with a Mutant CCT5 Subunit: Clues for Future Developments to Improve Differential Diagnosis and Personalized Therapy.

Scalia F, de Macario E, Bonaventura G, Cappello F, Macario A Biology (Basel). 2023; 12(5).

PMID: 37237456 PMC: 10215455. DOI: 10.3390/biology12050641.

Structural and Dynamic Disturbances Revealed by Molecular Dynamics Simulations Predict the Impact on Function of CCT5 Chaperonin Mutations Associated with Rare Severe Distal Neuropathies.

Scalia F, Lo Bosco G, Paladino L, Vitale A, Noori L, de Macario E Int J Mol Sci. 2023; 24(3).

PMID: 36768350 PMC: 9917133. DOI: 10.3390/ijms24032018.

References

Thulasiraman V, Ferreyra R, Frydman J . Folding assays. Assessing the native conformation of proteins. Methods Mol Biol. 2001; 140:169-77. DOI: 10.1385/1-59259-061-6:169. View

Pappenberger G, McCormack E, Willison K . Quantitative actin folding reactions using yeast CCT purified via an internal tag in the CCT3/gamma subunit. J Mol Biol. 2006; 360(2):484-96. DOI: 10.1016/j.jmb.2006.05.003. View

Roobol A, Carden M . Subunits of the eukaryotic cytosolic chaperonin CCT do not always behave as components of a uniform hetero-oligomeric particle. Eur J Cell Biol. 1999; 78(1):21-32. DOI: 10.1016/S0171-9335(99)80004-1. View

Knee K, Goulet D, Zhang J, Chen B, Chiu W, King J . The group II chaperonin Mm-Cpn binds and refolds human γD crystallin. Protein Sci. 2010; 20(1):30-41. PMC: 3047059. DOI: 10.1002/pro.531. View

Frydman J . Folding of newly translated proteins in vivo: the role of molecular chaperones. Annu Rev Biochem. 2001; 70:603-47. DOI: 10.1146/annurev.biochem.70.1.603. View

Spiess C, Miller E, McClellan A, Frydman J . Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins. Mol Cell. 2006; 24(1):25-37. PMC: 3339573. DOI: 10.1016/j.molcel.2006.09.003. View

Kim S, Willison K, Horwich A . Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains. Trends Biochem Sci. 1994; 19(12):543-8. DOI: 10.1016/0968-0004(94)90058-2. View

Flaugh S, Kosinski-Collins M, King J . Contributions of hydrophobic domain interface interactions to the folding and stability of human gammaD-crystallin. Protein Sci. 2005; 14(3):569-81. PMC: 2279286. DOI: 10.1110/ps.041111405. View

Thulasiraman V, Yang C, Frydman J . In vivo newly translated polypeptides are sequestered in a protected folding environment. EMBO J. 1999; 18(1):85-95. PMC: 1171105. DOI: 10.1093/emboj/18.1.85. View

10.

Kosinski-Collins M, King J . In vitro unfolding, refolding, and polymerization of human gammaD crystallin, a protein involved in cataract formation. Protein Sci. 2003; 12(3):480-90. PMC: 2312441. DOI: 10.1110/ps.0225503. View

11.

Frydman J, Nimmesgern E, Erdjument-Bromage H, Wall J, Tempst P, Hartl F . Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits. EMBO J. 1992; 11(13):4767-78. PMC: 556952. DOI: 10.1002/j.1460-2075.1992.tb05582.x. View

12.

Scheres S, Chen S . Prevention of overfitting in cryo-EM structure determination. Nat Methods. 2012; 9(9):853-4. PMC: 4912033. DOI: 10.1038/nmeth.2115. View

13.

Hartl F, Bracher A, Hayer-Hartl M . Molecular chaperones in protein folding and proteostasis. Nature. 2011; 475(7356):324-32. DOI: 10.1038/nature10317. View

14.

Dekker C, Roe S, McCormack E, Beuron F, Pearl L, Willison K . The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins. EMBO J. 2011; 30(15):3078-90. PMC: 3160183. DOI: 10.1038/emboj.2011.208. View

15.

Llorca O, Martin-Benito J, Grantham J, Hynes G, Willison K, Carrascosa J . Eukaryotic chaperonin CCT stabilizes actin and tubulin folding intermediates in open quasi-native conformations. EMBO J. 2000; 19(22):5971-9. PMC: 305829. DOI: 10.1093/emboj/19.22.5971. View

16.

Neirynck K, Waterschoot D, Vandekerckhove J, Ampe C, Rommelaere H . Actin interacts with CCT via discrete binding sites: a binding transition-release model for CCT-mediated actin folding. J Mol Biol. 2005; 355(1):124-38. DOI: 10.1016/j.jmb.2005.10.051. View

17.

Bigotti M, Clarke A . Chaperonins: The hunt for the Group II mechanism. Arch Biochem Biophys. 2008; 474(2):331-9. DOI: 10.1016/j.abb.2008.03.015. View

18.

Llorca O, Martin-Benito J, Gomez-Puertas P, Willison K, Carrascosa J, Valpuesta J . Analysis of the interaction between the eukaryotic chaperonin CCT and its substrates actin and tubulin. J Struct Biol. 2001; 135(2):205-18. DOI: 10.1006/jsbi.2001.4359. View

19.

Zhang Q, Yu D, Seo S, Stone E, Sheffield V . Intrinsic protein-protein interaction-mediated and chaperonin-assisted sequential assembly of stable bardet-biedl syndrome protein complex, the BBSome. J Biol Chem. 2012; 287(24):20625-35. PMC: 3370246. DOI: 10.1074/jbc.M112.341487. View

20.

Tang G, Peng L, Baldwin P, Mann D, Jiang W, Rees I . EMAN2: an extensible image processing suite for electron microscopy. J Struct Biol. 2006; 157(1):38-46. DOI: 10.1016/j.jsb.2006.05.009. View