The 3.6 Å cryo-em structure of the outer heptameric α-ring of human 26s immunoproteasome in the preactivation state

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Abstract

The 26S proteasome is a unique multicatalytic proteinase complex, together with a ubiquitination system, providing controlled degradation of most intracellular eukaryotic proteins. The problem of studying the proteasome is the multiplicity of its intracellular forms, which are formed due to the modularity of the proteasome assembly process. In this study, using cryoelectron microscopy, we described for the first time the structure of the 26S human immunoproteasome in comparison with its constitutive form with a resolution of 3.6 Å. A detailed analysis of the structural features of the two complexes revealed the opening of the entrance in the outer heptameric 20S ring of the immunoproteasome subunit due to the separation of the N-terminal regions of the PSMA4 and PSMA5 subunits and the formation of a π–π stacking between the amino acid residues Tyr5 and Phe9 of the PSMA5 and PSMA6 subunits, respectively. The revealed removal of steric obstruction in the central channel of the 20S subunit may indicate the preactivation phenotype of the 26S human immunoproteasome, even in the absence of a bound substrate.

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About the authors

G. A. Saratov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: belogurov@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

T. N. Baymukhametov

National Research Center, “Kurchatov Institute”

Email: belogurov@ibch.ru
Russian Federation, pl. Academica Kurchatova 1, Moscow, 123182

A. L. Konevega

National Research Center, “Kurchatov Institute”; Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre, Kurchatov Institute; Institute of Biomedical Systems and Biotechnologies, Peter the Great St. Petersburg Polytechnic University

Email: belogurov@ibch.ru
Russian Federation, pl. Academica Kurchatova 1, Moscow, 123182; mcr. Orlova Roshcha 1, Gatchina, 188300; ul. Politekhnicheskaya 29, St. Petersburg, 195251

А. A. Kudriaeva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: belogurov@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

А. А. Belogurov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS; Evdokimov Moscow State University of Medicine and Dentistry

Author for correspondence.
Email: belogurov@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997; ul. Delegatskaya 20/1, Moscow, 127473

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Supplementary files

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2. Fig. 1. (a) – Representative initial cryo-EM image of proteasome subunits of constitutive (c26S) and immune (i26S) forms; (b) – result of two-dimensional classification of the initial set of projections; (c) – subsamples of 26S proteasome subunits projections.

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3. Fig. 2. Heterogeneous Refinement result for the constitutive c26S (a) and immune i26S (b) forms of the proteasome. Orthogonal sections of cryo-EM maps along three axes for three structural organizations of proteasome subunits are shown. Absolute numbers of the corresponding projections in the data set after two-dimensional classification are indicated.

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4. Fig. 3. Correlation curves (Fourier Shell Correlation, FSC) for the constitutive c26S (a) and immune i26S (b) forms of the proteasome subunit. Shown are curves for local refinement of the 19S subunit (19S local, blue), local refinement of the core 20S subunit (20S local, orange), global refinement of the 26S subunit aligned with 20S (26S global, green), and refinement using the 3D Flexible Refinement method (26S 3Dflex, red). The dotted lines indicate two standard correlation thresholds of 0.5 and 0.143.

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5. Fig. 4. The result of the local resolution analysis for the constitutive c26S (a) and immune i26S (b) forms of the proteasome subunit, as well as the distribution of 26S projections according to the found angular orientations. Cryo-EM maps obtained with 3D Flexible Refinement and additionally processed with DeepEMhancer were used to visualize the local resolution. A lateral view, a central slice, and a top view are shown.

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6. Fig. 5. Comparative analysis of the global structure of the human 26S proteasome in constitutive (c26S) and immune (i26S) forms. The structure of the human 26S proteasome PDB 5GJR is given as a reference.

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7. Fig. 6. (a) – Analysis of electron density in the substrate-binding pockets of the hexameric ATPase ring; (b) – anchoring of HbYX motifs of Rpt3 and Rpt6 subunits in hydrophobic cavities of the heptameric α-ring; (c) – presence of immunosubunits in the proteasome isolated from HeLa cells pre-treated with IFNγ, based on changes in electron density at the positions of characteristic amino acid residues of the proteasome of the immune phenotype.

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8. Fig. 7. Comparative analysis of the proximal (directly interacting with the 19S regulatory subunit) and axial (remote) heptameric α-ring (a) of the N-terminal regions of α-subunits in the structure of the immunoproteasome in comparison with the proteasome of the constitutive phenotype (b); (c) – cooperative shift of the N-terminal fragments of the PSMA1, PSMA4 and PSMA6 subunits in the direction of the PSMA2 subunit.

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9. Fig. 8. (a) – Visualization of the electron density of the proximal α-ring in the region of the entrance to the inner chamber of the catalytic 20S subunit; (b) – dissociation of the system of ionic bonds observed in the case of the constitutive proteasome, between the amino acid residues Arg5 PSMA7 and Asp9 PSMA5, Arg10 PSMA5 and Asp7 PSMA1, as well as Asn8 PSMA1 and Arg3 PSMA4 in the structure of the immunoproteasome; (c) – formation of the π-π-fold between the residues Tyr5 and Phe9 of the PSMA4 and PSMA6 subunits.

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