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bioRxiv · 10.1101/2025.01.30.635686

Characterization of conformational dynamics and allostery of the catalytic domain of human mitochondrial YME1L protease

Black, M. K.·Kim, A. S.·Chen, C. Y.·Goncalves, M. M.·Waseem, S.·Vahidi, S.·Huang, R.
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Abstract

Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L protease is an important contributor to proteostasis which belongs to the AAA+ (ATPases Associated with diverse cellular Activities) protease family and is anchored to the inner mitochondrial membrane. YME1L plays a pivotal role in mitochondrial protein quality control by selectively degrading misfolded and native proteins. The precise mechanisms by which nucleotide binding and hydrolysis influence YME1Ls conformational dynamics, proteolytic activity, and stability remain unclear. Here we characterize the conformational dynamics and allosteric regulation of the YME1L catalytic domain. Using a hexameric soluble YME1L construct, we employ hydrogen/deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) spectroscopy to demonstrate that nucleotide binding reduces the backbone flexibility and modulates the side-chain dynamics of the AAA+ domain, while Zn{superscript 2} binding stabilizes the protease domain. We also reveal novel long-range allostery between the AAA+ and protease domains of YME1L, mediated by a critical salt bridge on the inter-domain interface. We show the importance of the salt bridge in facilitating ATP-dependent substrate degradation by YME1L. Additionally, we show that ATP binding stabilizes the structure of the catalytic domain of YME1L and protects it from chemical modification- and heat-induced aggregation. These findings explain the nucleotide-driven regulation of YME1L and provide novel insights into understanding its proteolytic activity and structural stability under physiological and stress conditions.

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Black, M. K., Kim, A. S., Chen, C. Y., Goncalves, M. M., Waseem, S., Vahidi, S., Huang, R.. 2025-01-30. Characterization of conformational dynamics and allostery of the catalytic domain of human mitochondrial YME1L protease. https://doi.org/10.1101/2025.01.30.635686

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