The Structural Dynamics of IRE1 and its Interaction with Unfolded Peptides
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Curated by eLife
eLife Assessment
In this important study, the authors conducted atomistic molecular dynamics simulations to probe the interactions between IRE and unfolded peptides. The results help reconcile contradicting experimental findings in the literature and offer mechanistic insights into the activation of the unfolded protein response. The level of evidence is considered solid, although the use of enhanced sampling and more quantitative analysis would further strengthen the conclusions.
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Abstract
Abstract
The unfolded protein response (UPR) is a crucial signaling network that preserves endoplasmic reticulum (ER) homeostasis, impacting both health and disease. When ER stress occurs, often due to an accumulation of unfolded proteins in the ER lumen, the UPR initiates a broad cellular program to counteract cytotoxic effects. Inositol-requiring enzyme 1 (IRE1), a conserved ER-bound protein, is a key sensor of ER stress and activator of the UPR. While biochemical studies confirm IRE1’s role in recognizing unfolded polypeptides, high-resolution structures showing direct interactions remain elusive. Consequently, the precise structural mechanism by which IRE1 senses unfolded proteins is debated. In this study, we employed advanced molecular modeling and extensive atomistic molecular dynamics simulations to clarify how IRE1 detects unfolded proteins. Our results demonstrate that IRE1’s luminal domain directly interacts with unfolded peptides and reveal how these interactions can stabilize higher-order oligomers. We provide a detailed molecular characterization of unfolded peptide binding, identifying two distinct binding pockets at the dimer’s center, separate from its central groove. Furthermore, we present high-resolution structures illustrating how BiP associates with IRE1’s oligomeric interface, thus preventing the formation of larger complexes. Our structural model reconciles seemingly contradictory experimental findings, offering a unified perspective on the diverse sensing models proposed. Ultimately, we elucidate the structural dynamics of unfolded protein sensing by IRE1, providing key insights into the initial activation of the UPR.
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eLife Assessment
In this important study, the authors conducted atomistic molecular dynamics simulations to probe the interactions between IRE and unfolded peptides. The results help reconcile contradicting experimental findings in the literature and offer mechanistic insights into the activation of the unfolded protein response. The level of evidence is considered solid, although the use of enhanced sampling and more quantitative analysis would further strengthen the conclusions.
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Reviewer #1 (Public review):
Summary:
This work provides structural and mechanistic insights into the disordered protein recognition process inside the endoplasmic reticulum by the inositol-requiring enzyme 1. Using state-of-the-art molecular dynamics simulation tools, the authors propose a mechanism of disordered protein recognition that reconciles contradictory findings of biochemical and structural biology experiments.
Strengths:
(1) All MD simulations have been carried out in triplicate, and several different folded conformations were generated using alphafold2. This provides adequate statistics to draw meaningful conclusions from the simulations.
(2) Potential limitations of the disordered protein force fields and water models have been taken into consideration. Particularly, performing the simulation in both TIP3P and TIP4PD water …
Reviewer #1 (Public review):
Summary:
This work provides structural and mechanistic insights into the disordered protein recognition process inside the endoplasmic reticulum by the inositol-requiring enzyme 1. Using state-of-the-art molecular dynamics simulation tools, the authors propose a mechanism of disordered protein recognition that reconciles contradictory findings of biochemical and structural biology experiments.
Strengths:
(1) All MD simulations have been carried out in triplicate, and several different folded conformations were generated using alphafold2. This provides adequate statistics to draw meaningful conclusions from the simulations.
(2) Potential limitations of the disordered protein force fields and water models have been taken into consideration. Particularly, performing the simulation in both TIP3P and TIP4PD water models ensures that the conclusions drawn are not influenced by the force field choice.
(3) The binding of a large number of disordered peptides was investigated, ensuring that the conclusions drawn about disordered peptide recognition are sufficiently general.
Weaknesses:
(1) The timescales of the peptide recognition and unbinding process are much longer than what can be sampled from unbiased simulations. Therefore, the proposed mechanism of recognition should only be considered a hypothesis based on the results presented here. For example, peptides that do not dissociate within one one-microsecond MD simulation are considered to be stable binders. However, they may not have a viable way to bind to the narrow protein cleft in the first place.
(2) Oftentimes, representative structures sampled from MD simulation are used to draw conclusions (e.g., Figure 4 about the role of R161 mutation in binding affinity). This is not appropriate as one unbinding event being observed or not observed in a microsecond-long trajectory does not provide sufficient information about the binding strength of the free energy difference.
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Reviewer #2 (Public review):
Summary:
In this manuscript, the authors investigated the interactions between IRE and unfolded peptides using all-atom molecular dynamics simulations. The interactions between a couple of unfolded peptides and IRE might shed light on the activation of the UPR.
Strengths:
(1) Well-written manuscript tailored for a biology audience.
(2) State-of-the-art structural predictions and all-atom simulations.
(3) Validation with existing experimental data
(4) Clear schematic diagram summarizing the mechanisms learned from simulations.
(5) Shared simulation data and code in a public repository.
Weaknesses:
(1) Improving presentation to include more computational details.
(2) More quantitative analysis in addition to visual structures.
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Reviewer #3 (Public review):
Summary:
In this important work, the authors use extensive MD simulations to study how the IRE1 protein can detect unfolded peptides. Their study consolidates contradicting experimental results and offers a unique view of the different sensing models that have been proposed in the literature. Overall, it is an excellent study that is quite extensive. The research is solid, meticulous, and carefully performed, leading to convincing conclusions.
Strengths:
The strength of this work is the extensive and meticulous molecular dynamics simulations. The authors use and investigate different structural models, for example, carefully comparing a model based on a PDB structure with reconstructed loops with an AlphaFold 2 Multimer model. The author also investigates a wide range of different protein structural models …
Reviewer #3 (Public review):
Summary:
In this important work, the authors use extensive MD simulations to study how the IRE1 protein can detect unfolded peptides. Their study consolidates contradicting experimental results and offers a unique view of the different sensing models that have been proposed in the literature. Overall, it is an excellent study that is quite extensive. The research is solid, meticulous, and carefully performed, leading to convincing conclusions.
Strengths:
The strength of this work is the extensive and meticulous molecular dynamics simulations. The authors use and investigate different structural models, for example, carefully comparing a model based on a PDB structure with reconstructed loops with an AlphaFold 2 Multimer model. The author also investigates a wide range of different protein structural models that probe different aspects of the peptide sensing process. These solid and meticulous MD simulations allow the authors to obtain convincing conclusions concerning the peptide sensing process of the IRE1 protein.
Weaknesses:
A potential weakness of the study is the usage of equilibrium (unbiased) molecular dynamics simulations, so that processes and conformational changes on the microsecond time scale can be probed. Furthermore, there can be inaccuracies and biases in the description of unfolded peptides and protein segments due to the protein force fields. Here, it should be noted that the authors do acknowledge these possible limitations of their study in the conclusions.
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