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bioRxiv · 10.1101/2024.12.30.630732

Characterization and modulation of human insulin degrading enzyme conformational dynamics to control enzyme activity

Mancl, J. M.·Liang, w. G.·bayhi, N. L.·Wei, H.·Carragher, B.·Potter, C. S.·Tang, W.-J.
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Abstract

Insulin degrading enzyme (IDE) is a dimeric M16A zinc metalloprotease that degrades amyloidogenic peptides diverse in shape and sequence, including insulin and amyloid-{beta}, to prevent toxic amyloid fibril formation. IDE has a hollow catalytic chamber formed by 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 a new substrate. Here, we present six cryo-EM structures of the IDE dimer at 3.0-5.1 [A] resolution, obtained in the presence of a sub-saturating concentration of insulin. Combining cryo-EM heterogeneity analysis with all-atom molecular dynamics (MD) simulations, we identified the structural basis and key residues for IDE conformational dynamics that were not previously revealed by IDE static structures. Notably R668 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 small-angle X-ray scattering analysis and enzymatic assays of an R668A mutant indicate a profound alteration of conformational dynamics and catalytic activity. By integrating coarse-grained MD simulations, our analysis reveals that IDE unfolds its substrates through the coordinated motion between IDE-N and IDE-C, as well as {beta}-sheet formation between IDE and insulin. Additionally, our time-resolved cryo-EM analysis uncovers IDE allostery within the IDE dimer. Collectively, our findings demonstrate the strength of combining experimental and computational approaches to probe protein dynamics and pave the way for developing substrate-specific modulators of IDE activity.

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Mancl, J. M., Liang, w. G., bayhi, N. L., Wei, H., Carragher, B., Potter, C. S., Tang, W.-J.. 2024-12-30. Characterization and modulation of human insulin degrading enzyme conformational dynamics to control enzyme activity. https://doi.org/10.1101/2024.12.30.630732

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