4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Yao Cong; Matthew L Baker; Joanita Jakana; David Woolford; Erik J Miller; Stefanie Reissmann; Ramya N Kumar; Alyssa M Redding-Johanson; Tanveer S Batth; Aindrila Mukhopadhyay; Steven J Ludtke; Judith Frydman; Wah Chiu

doi:10.1073/pnas.0913774107

4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Yao Cong, Matthew L Baker, Joanita Jakana, David Woolford, Erik J Miller, Stefanie Reissmann, Ramya N Kumar, Alyssa M Redding-Johanson, Tanveer S Batth, Aindrila Mukhopadhyay, Steven J Ludtke, Judith Frydman, Wah Chiu

121 Citationer (Scopus)

Abstract

The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ″5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

Originalsprog	Engelsk
Tidsskrift	Proceedings of the National Academy of Sciences of the United States of America
Vol/bind	107
Udgave nummer	11
Sider (fra-til)	4967-72
Antal sider	6
ISSN	0027-8424
DOI	https://doi.org/10.1073/pnas.0913774107
Status	Udgivet - 16 mar. 2010

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10.1073/pnas.0913774107

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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. (2010). 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. Proceedings of the National Academy of Sciences of the United States of America, 107(11), 4967-72. https://doi.org/10.1073/pnas.0913774107

4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement. / Cong, Yao; Baker, Matthew L; Jakana, Joanita et al.
I: Proceedings of the National Academy of Sciences of the United States of America, Bind 107, Nr. 11, 16.03.2010, s. 4967-72.

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

Cong, Y, Baker, ML, Jakana, J, Woolford, D, Miller, EJ, Reissmann, S, Kumar, RN, Redding-Johanson, AM, Batth, TS, Mukhopadhyay, A, Ludtke, SJ, Frydman, J & Chiu, W 2010, '4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement', Proceedings of the National Academy of Sciences of the United States of America, bind 107, nr. 11, s. 4967-72. https://doi.org/10.1073/pnas.0913774107

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abstract = "The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ″5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-{\AA} resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-{\AA} resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.",

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author = "Yao Cong and Baker, {Matthew L} and Joanita Jakana and David Woolford and Miller, {Erik J} and Stefanie Reissmann and Kumar, {Ramya N} and Redding-Johanson, {Alyssa M} and Batth, {Tanveer S} and Aindrila Mukhopadhyay and Ludtke, {Steven J} and Judith Frydman and Wah Chiu",

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T1 - 4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

AU - Cong, Yao

AU - Baker, Matthew L

AU - Jakana, Joanita

AU - Woolford, David

AU - Miller, Erik J

AU - Reissmann, Stefanie

AU - Kumar, Ramya N

AU - Redding-Johanson, Alyssa M

AU - Batth, Tanveer S

AU - Mukhopadhyay, Aindrila

AU - Ludtke, Steven J

AU - Frydman, Judith

AU - Chiu, Wah

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N2 - The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ″5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

AB - The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of ″5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique heterooligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7-Å resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-Å resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a Cα backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed ∼95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.

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KW - Animals

KW - Cattle

KW - Chaperonin Containing TCP-1/chemistry

KW - Cryoelectron Microscopy

KW - Crystallography, X-Ray

KW - Models, Molecular

KW - Molecular Sequence Data

KW - Protein Structure, Secondary

KW - Protein Subunits/chemistry

KW - Reproducibility of Results

KW - Static Electricity

KW - Surface Properties

U2 - 10.1073/pnas.0913774107

DO - 10.1073/pnas.0913774107

M3 - Journal article

C2 - 20194787

SN - 0027-8424

VL - 107

SP - 4967

EP - 4972

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 11

ER -

4.0-A resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement

Abstract

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