An empirical energy landscape reveals mechanism of proteasome in polypeptide translocation
- Department of Systems Biology, Harvard Medical School, United States
Figures
Figure 1 with 2 supplements
The architectures of the proteasomal ATPase complex and its interaction with substrate.
(A) A schematic showing a half 26S proteasome engaged with an unfolded substrate through the PL1s (color loops) on the ATPase subunits with bound nucleotides (color blobs). The disengaged PL1 is …
Figure 1—figure supplement 1
Schematics showing the key structural features and the nucleotide-binding status of the substrate-engaged proteasome identified in a cryo-EM study.
Each ATPase subunit is arranged vertically according to the order, or the approximate distance, of its pore-1 loop to the 20S core particle. An open interface is represented by a large gap. The …
Figure 1—figure supplement 2
Comparison between open and closed interfaces of ATPases on proteasome.
(A) The closed interface of the ATPase domains of Rpt3–Rpt4 in the ED1 cryo-EM state. Green: Rpt3; Cyan: Rpt4; Red: ATP; Arginine fingers on Rpt4 were shown with sticks. (B) The open interface of …
Figure 2 with 2 supplements
Parameterization of the nucleotide-dependent free-energy landscape.
(A) The interaction map of the residues on Rpt4 and Rpt3 with the bound ATP or ADP in the ED1 or ED2 states. Red: cis-interacting residues on Rpt3. Blue: trans-interacting residues on Rpt4. (B) A …
Figure 2—figure supplement 1
Phosphate groups interact weakly with the proteasomal ATPases.
The degradation rate of ubiquitylated cyclinB-iRFP was measured at different phosphate concentrations, normalized by the rate at zero phosphate. The red curve is a fitting using the inhibitor …
Figure 2—figure supplement 2
Root-mean-square deviation (RMSD) of the nucleotide-interacting residues in the cis pockets among different proteasomal states.
The RMSD of the nucleotide-interacting residues in the cis binding pockets among different proteasomal states was analyzed as described in 'Analysis of cryo-EM structures'. The first three …
Figure 3 with 2 supplements
Evaluation of the FEL model parameters.
(A) A schematic showing three categories of nucleotide pockets with their corresponding dissociation constants Kd. (B) Single-molecule nucleotide-proteasome interaction assay. 200 nM Alexa647-ATP …
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Figure 3—source data 1
Degradation kinetics of ubiquitylated cyclinB-iRFP at various concentrations of substrate and proteasome.
Related to Figure 3E.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig3-data1-v2.pdf
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Figure 3—source data 2
Degradation kinetics of ubiquitylated cyclinB-iRFP in the presence of 500 µM ATP and various concentrations of ADP.
Related to Figure 3F.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig3-data2-v2.pdf
Figure 3—figure supplement 1
A block diagram for simulating the dynamics of proteasome using the FEL model.
See Materials and methods 'Constructing the nucleotide-dependent free-energy landscape (FEL) of the ATPase complex on proteasome to simulate its confirmational dynamics' for a detailed description …
Figure 3—figure supplement 2
Examples of single-molecule traces showing processive ubiquitin chain removal.
Purified cycB_NT(K18,36,64) was ubiquitylated by APC-Cdh1 and E2 UbcH10 with Dylight550-Ub and was subjected to single-molecule proteasome assay as described in Materials and methods …
Figure 4 with 5 supplements
Evaluating the FEL-predicted degradation kinetics.
(A) Examples of simulated kinetics of translocation on individual proteasome particles under indicated nucleotide conditions. (B) The translocation rate of cyclinB-iRFP measured at various …
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Figure 4—source data 1
Degradation kinetics of ubiquitylated cyclinB-iRFP in the presence of various concentrations of ATP.
Related to Figure 4B.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig4-data1-v2.pdf
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Figure 4—source data 2
Degradation kinetics of ubiquitylated cyclinB-iRFP in the presence of various concentrations of ATP.
Related to Figure 4B.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig4-data2-v2.pdf
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Figure 4—source data 3
Degradation kinetics of ubiquitylated cyclinB-iRFP in the presence of 500 µM ATP and various concentrations of ATP-γS.
Related to Figure 4C and D.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig4-data3-v2.pdf
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Figure 4—source data 4
Degradation kinetics of ubiquitylated cyclinB-iRFP in the presence of 15 mM ATP and various concentrations of ATP-γS.
Related to Figure 4D.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig4-data4-v2.pdf
Figure 4—figure supplement 1
The workflow of this study, including the observations used for model construction, experimental validations of the simulated ATPase dynamics, and the major insights into the ATPase mechanism.
Figure 4—figure supplement 2
High ATP concentration does not cause proteasome disassembly.
Purified human 26S proteasome was incubated with ATP-Mg2+ at indicated concentrations. After incubation, the sample was fixed with 1 mM BS3 (bis(sulfosuccinimidyl)suberate) for 30 min and was …
Figure 4—figure supplement 3
High concentration of Mg2+ alone does not affect the degradation kinetics.
The quantitative degradation assay of ubiquitylated cyclinB-iRFP was performed as described in Materials and methods with 0.5 mM ATP, with or without an extra 15 mM MgCl2. Two substrate …
Figure 4—figure supplement 4
High ATP concentration does not lead to partial protein degradation or uncoupling between ubiquitylation and degradation.
Ubiquitylated cyclinB-iRFP was radiolabeled with (Lyubimov et al., 2011) p at both the N- and C-terminus using protein kinase A. The degradation assay was performed as described in methods and was …
Figure 4—figure supplement 5
High ATP concentration does not inhibit the ATPase activity of proteasome.
The rate of ATP hydrolysis of purified 26S proteasome was measured in the presence of varying concentrations of ATP-Mg2+ using a Malachite green assay (Peth et al., 2013b). Denatured ovalbumin was …
Figure 5 with 5 supplements
Evaluating the FEL predictions for structurally-stable substrates.
(A) The initial degradation rate of ubiquitylated cyclinB-DHFR-iRFP by purified 26S proteasome with or without 0.8 mM folic acid (FA), overlaid with the FEL model prediction. The inset shows the …
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Figure 5—source data 1
Degradation kinetics of ubiquitylated cyclinB-DHFR-iRFP in the presence of various concentrations of ATP without folic acid.
Related to Figure 5A and B.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig5-data1-v2.pdf
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Figure 5—source data 2
Degradation kinetics of ubiquitylated cyclinB-DHFR-iRFP in the presence of various concentrations of ATP with folic acid.
Related to Figure 5A and B.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig5-data2-v2.pdf
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Figure 5—source data 3
Degradation kinetics of ubiquitylated cyclinB-iRFP in the presence of cyclinB-DHFR-iRFPdark as competitor, with either ADP or ATP-γS in the buffer.
Related to Figure 5F.
- https://cdn.elifesciences.org/articles/71911/elife-71911-fig5-data3-v2.pdf
Figure 5—figure supplement 1
Folic acid slows down the degradation of cyclinB-DHFR-iRFP, but not cyclinB-iRFP.
100 nM ubiquitylated cyclinB-DHFR-iRFP was tested in the quantitative degradation assay with 2 nM proteasome at various concentrations of folic acid (A) or methotrexate (C) as indicated in the …
Figure 5—figure supplement 2
CyclinB-DHFR-iRFP was completely degraded by the proteasome in the presence of folic acid.
Ubiquitylated cyclinB-DHFR-iRFP was radiolabeled with (Lyubimov et al., 2011) p at both the N- and C-terminus using protein kinase A and was subject to degradation by 2 nM purified human 26S …
Figure 5—figure supplement 3
The degradation rate of cyclinB-DHFR-iRFP is inversely proportional to the folic acid concentrations.
The degradation rate of cyclinB-DHFR-iRFP was measured at various folic acid concentrations as described in the Methods. Its inverse was plotted against the folic acid concentration.
Figure 5—figure supplement 4
The rate of ATP hydrolysis versus ATP concentration as predicted by the FEL model.
The predicted rate of ATP hydrolysis per ATPase hexamer was plotted against the concentration of ATP in the FEL simulation as described in Materials and methods 'Monte Carlo simulation of the FEL …
Figure 5—figure supplement 5
Sensitivity of the simulated translocation rates to parameter variations.
A parameter is either increased or decreased by 30%, except for kon which is varied by threefold due to the large uncertainty in its estimation. The simulated translocation rates under various …
Figure 6 with 6 supplements
Organization of proteasomal conformations in dynamical space.
(A) A diagram showing the steady-state transitions between the ATPase complex conformations in the FEL model. Each node represents a unique hexamer conformation, whose size is in proportion to its …
Figure 6—figure supplement 1
The rates of substrate translocation and ATP hydrolysis for a Walker-B mutant proteasome.
The translocation rate and ATPase activity were calculated using the FEL model in which Rpt3’s ATP hydrolysis activity was abolished (WBM). The results are compared with those of the wild-type (WT) …
Figure 6—figure supplement 2
The typical sequence of elementary steps underlying the ED1-to-ED2 state transition.
In a simulation of the FEL model, the probabilities of all the pathways that lead to the ED1 to ED2 transition were calculated. The pathway with the highest probability is presented. This pathway is …
Figure 6—figure supplement 3
The typical sequence of elementary steps underlying the ED1-to-EC state transitions.
The same analysis was performed as in Figure 6—figure supplement 2, but for the ED1 to EC transition.
Figure 6—figure supplement 4
The Lid-ATPase interaction may account for the observed dissymmetry in conformational occupancies in previous cryo-EM studies.
In an FEL model simulation, the standard free energy of the ED-like conformation (gray) which mimics the ATPase architecture in the EA-like states in the cryo-EM studies was lowered by an arbitrary …
Figure 6—figure supplement 5
The Lid-ATPase interaction may account for the different growth phenotypes of yeast Walker-B mutants.
The translocation rate was calculated for each Walker-B mutant proteasome (ATP hydrolysis set to zero) using the FEL model involving the ATPase-Lid interaction as described in Materials and methods …
Figure 6—figure supplement 6
Global dynamical space with heterogeneous ATPase parameters.
In the simulation, we let the basal energy Ebi of the ith ATPase = α×δi. δi is a random number from 0 to 1 and α is a scaling constant. In (A), we varied the value of α while fixing the ratios of Ebi…
Author response image 1
Testing alternative FEL models.
(A) Steady-state occupancies for different conformations. See text for the definition of each model. B-D. Comparison of different models in predicting translocation rates in various nucleotide …
Author response image 2
Global dynamical space in the presence of variations in e_b.
(A) Varying the amplitude α of e_b variation while fixing the ratios. In this example, δi=(0.45,0.15,0.83,1.0, 0.68,0.88). B. Two cases with alternative δi and the same amplitude. C. The same e_b …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pT7-CyclnB-iRFP (plasmid) | This study | pLM254 | For expressing cyclinB-iRFP(available upon request) |
| Recombinant DNA reagent | pT7-cyclinB-DHFR-iRFP (plasmid) | This study | pLM428 | For expressing cyclinB-DHFR-iRFP(available upon request) |
| Recombinant DNA reagent | pT7-cyclinB-iRFP-DHFR (plasmid) | This study | pLM429 | For expressing cyclinB-iRFP-DHFR(available upon request) |
| Recombinant DNA reagent | pT7-cyclinB (K18, 36, 64) (plasmid) | This study | pLM120 | For expressing cyclinB (K18, 36, 64)(available upon request) |
| Peptide, recombinant protein | Dylight550-Ubiquitin | Lu et al., 2015; Puchades et al., 2020 | Dy550-Ub | Available upon request |
| Peptide, recombinant protein | Human 26S proteasome | HEK293 cell (Rpn11-HTBH) | hPTSM | |
| Peptide, recombinant protein | Human 26S proteasome SNAP-Rpn3 | HE293 cell (SNAP-Rpn3) This study | hPTSM-SNAP | |
| Cell line (Human) | HEK293 | Lab stock (commonly available) | HEK293 | |
| Cell line (Human) | HEK293-SNAP-Rpn3 | This study | HEK293-Rpn3-SNAP | For expressing SNAP-Rpn3 proteasome |
| Chemical compound, drug | ATP-gS | Sigma-Aldrich | A1388 | |
| Chemical compound, drug | A647-ATP | Thermo Fisher Scientific | A22362 | |
| Chemical compound, drug | Folic acid | Sigma-Aldrich | F8758 | |
| Chemical compound, drug | Methotrexate | Sigma-Aldrich | A6770 | |
| Chemical compound, drug | Biliverdin | Sigma-Aldrich | 30891 | |
| Chemical compound, drug | SNAP-surface-549 | NEB | S9112S | |
| Strain, strain background (Escherichia coli) | NiCo21 DE3 | NEB | C2529H | |
| Software, algorithm | MATLAB 2018 | MathWorks | ||
| Software, algorithm | Pajek | Pajek | http://vlado.fmf.uni-lj.si/pub/networks/pajek/ | |
| Software, algorithm | Proteasome FEL model | This study | https://github.com/luyinghms/Proteasome-FEL-model.git; Ying, 2022 | Source code for the FEL model |
| Chemical compound, drug | ATP-gS | Sigma-Aldrich | A1388 |
Additional files
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Supplementary file 1
ATPase conformations and steady-state occupancies.
“Close interface”: the six digits indicate the Rpt6-Rpt3, Rpt3-Rpt4, Rpt4-Rpt5, Rpt5-Rpt1, Rpt1-Rpt2, Rpt2-Rpt6 interfaces. “0”: open; “1”: closed. “Engaged ATPases”: the six digits indicate Rpt6, Rpt3, Rpt4, Rpt5, Rpt1, Rpt2. “0”: disengaged subunit; “1”: engaged subunit. “PL1 registry”: the six digits indicate the PL1s on Rpt6, Rpt3, Rpt4, Rpt5, Rpt1, Rpt2. “1~5”: part of the staircase architecture. “1” is closest to the CP; and “5” is farthest from the CP. “7”: disengaged PL1 at the top registry. “Steady-state occupancy (%)”: the steady-state occupancy of each conformation in a FEL simulation. 2. Steady-state transition rates among ATPase conformations in the FEL model. “Total transition”: the total rate of transitions from conformation 1 to conformation two and reverse. Numbers are normalized by the highest value set to 100. “Net transition”: the absolute value of the rate difference between conf1-conf2 and conf2-conf1 transitions. Numbers are normalized by the same factor as above. “Translocation”: F: the net effect of conf1-conf2 transitions is a forward translocation of substrate; “B”: the net effect is a backward translocation; “N”: this conformational transition does not lead to translocation.
- https://cdn.elifesciences.org/articles/71911/elife-71911-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71911/elife-71911-transrepform1-v2.docx
