Characterization and modulation of human insulin degrading enzyme conformational dynamics to control enzyme activity
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eLife Assessment
The manuscript by Mancl et al. provides valuable mechanistic insights into the conformational dynamics of Insulin Degrading Enzyme (IDE), a zinc metalloprotease involved in the clearance of various bioactive peptides. Supported by a convincing combination of cryo-EM, SEC-SAXS, enzymatic assays, and molecular dynamics simulations, the study characterizes the dynamic transitions between IDE's open and closed states in the presence of a sub-saturating concentration of insulin. This work contributes to a refined model of IDE's functional cycle, enhancing our understanding of its role in proteolysis.
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Abstract
Insulin degrading enzyme (IDE) is a dimeric 110 kDa M16A zinc metalloprotease that degrades amyloidogenic peptides diverse in shape and sequence, including insulin, amylin, and amyloid-β, to prevent toxic amyloid fibril formation. IDE has a hollow catalytic chamber formed by four homologous subdomains organized into two ∼55 kDa N- and C-domains (IDE-N and IDE-C, respectively), in which peptides bind, unfold, and are repositioned for proteolysis. IDE is known to transition between a closed state, poised for catalysis, and an open state, able to release cleavage products and bind new substrate. Here, we present five cryoEM structures of the IDE dimer at 3.0-4.1 Å resolution, obtained in the presence of a sub-saturating concentration of insulin. Analysis of the heterogeneity within the particle populations comprising these structures combined with all-atom molecular dynamics (MD) simulations permitted a comprehensive characterization of IDE conformational dynamics. Our analysis identified the structural basis and key residues for these dynamics that were not revealed by IDE static structures. Notably arginine-668 serves as a molecular latch mediating the open-close transition and facilitates key protein motions through charge-swapping interactions at the IDE-N/C interface. Our size-exclusion chromatography-coupled small-angle X-ray scattering and enzymatic assays of an arginine-668 to alanine mutant indicate a profound alteration of conformational dynamics and catalytic activity. Taken together, this work highlights the power of integrating experimental and computational methodologies to understand protein dynamics, offers the molecular basis of unfoldase activity of IDE, and provides a new path forward towards the development of substrate-specific modulators of IDE activity.
Article activity feed
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eLife Assessment
The manuscript by Mancl et al. provides valuable mechanistic insights into the conformational dynamics of Insulin Degrading Enzyme (IDE), a zinc metalloprotease involved in the clearance of various bioactive peptides. Supported by a convincing combination of cryo-EM, SEC-SAXS, enzymatic assays, and molecular dynamics simulations, the study characterizes the dynamic transitions between IDE's open and closed states in the presence of a sub-saturating concentration of insulin. This work contributes to a refined model of IDE's functional cycle, enhancing our understanding of its role in proteolysis.
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Reviewer #1 (Public review):
Summary:
Mancl et al. present cryo-EM structures of the Insulin Degrading Enzyme (IDE) dimer and characterize its conformational dynamics by integrating structures with SEC-SAXS, enzymatic activity assays, and all-atom molecular dynamics (MD) simulations. They present five cryo-EM structures of the IDE dimer at 3.0-4.1 Å resolution, obtained with one of its substrates, insulin, added to IDE in a 1:2 ratio. The study identified R668 as a key residue mediating the open-close transition of IDE, a finding supported by simulations and experimental data. The work offers a refined model for how IDE recognizes and degrades amyloid peptides, incorporating the roles of IDE-N rotation and charge-swapping events at the IDE-N/C interface.
Strengths:
The study by Mancl et al. uses a combination of experimental (cryoEM, …
Reviewer #1 (Public review):
Summary:
Mancl et al. present cryo-EM structures of the Insulin Degrading Enzyme (IDE) dimer and characterize its conformational dynamics by integrating structures with SEC-SAXS, enzymatic activity assays, and all-atom molecular dynamics (MD) simulations. They present five cryo-EM structures of the IDE dimer at 3.0-4.1 Å resolution, obtained with one of its substrates, insulin, added to IDE in a 1:2 ratio. The study identified R668 as a key residue mediating the open-close transition of IDE, a finding supported by simulations and experimental data. The work offers a refined model for how IDE recognizes and degrades amyloid peptides, incorporating the roles of IDE-N rotation and charge-swapping events at the IDE-N/C interface.
Strengths:
The study by Mancl et al. uses a combination of experimental (cryoEM, SEC-SAXS, enzymatic assays) and computational (MD simulations, multibody analysis, 3DVA) techniques to provide a comprehensive characterization of IDE dynamics. The identification of R668 as a key residue mediating the open-to-close transition of IDE is a novel finding, supported by both simulations and experimental data presented in the manuscript. The work offers a refined model for how IDE recognizes and degrades amyloid peptides, incorporating the roles of IDE-N rotation and charge-swapping events at the IDE-N/C interface. The study identifies the structural basis and key residues for IDE dynamics that were not revealed by static structures.
Weaknesses:
Based on MD simulations and enzymatic assays of IDE, the authors claim that the R668A mutation in IDE affects the conformational dynamics governing the open-closed transition, which leads to altered substrate binding and catalysis. The functional importance of R668 would be substantiated by enzymatic assays that included some of the other known substrates of IDE than insulin such as amylin and glucagon.
It is unclear to what extent the force field (FF) employed in the MD simulations favors secondary structures and if the lack of any observed structural changes within the IDE domains in the simulations - which is taken to suggest that the domains behave as rigid bodies - stems from bias by the FF.
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Reviewer #2 (Public review):
Summary:
The manuscript describes various conformational states and structural dynamics of the Insulin degrading enzyme (IDE), a zinc metalloprotease by nature. Both open and closed-state structures of IDE have been previously solved using crystallography and cryo-EM which reveal a dimeric organization of IDE where each monomer is organized into N and C domains. C-domains form the interacting interface in the dimeric protein while the two N-domains are positioned on the outer sides of the core formed by C-domains. It remains elusive how the open state is converted into the closed state but it is generally accepted that it involves large-scale movement of N-domains relative to the C-domains. The authors here have used various complementary experimental techniques such as cryo-EM, SAXS, size-exclusion …
Reviewer #2 (Public review):
Summary:
The manuscript describes various conformational states and structural dynamics of the Insulin degrading enzyme (IDE), a zinc metalloprotease by nature. Both open and closed-state structures of IDE have been previously solved using crystallography and cryo-EM which reveal a dimeric organization of IDE where each monomer is organized into N and C domains. C-domains form the interacting interface in the dimeric protein while the two N-domains are positioned on the outer sides of the core formed by C-domains. It remains elusive how the open state is converted into the closed state but it is generally accepted that it involves large-scale movement of N-domains relative to the C-domains. The authors here have used various complementary experimental techniques such as cryo-EM, SAXS, size-exclusion chromatography, and enzymatic assays to characterize the structure and dynamics of IDE protein in the presence of substrate protein insulin whose density is captured in all the structures solved. The experimental structural data from cryo-EM suffered from a high degree of intrinsic motion among the different domains and consequently, the resultant structures were moderately resolved at 3-4.1 Å resolution. A total of five structures were generated by cryo-EM. The authors have extensively used Molecular dynamics simulation to fish out important inter-subunit contacts which involve R668, E381, D309, etc residues. In summary, authors have explored the conformational dynamics of IDE protein using experimental approaches which are complemented and analyzed in atomic details by using MD simulation studies. The studies are meticulously conducted and lay the ground for future exploration of the protease structure-function relationship.
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