Characterization of Conformational Dynamics and Structural Plasticity of the Catalytic Domain of Human Mitochondrial YME1L Protease

Mitochondrial proteostasis is essential to maintain cellular function and survival. YME1L is a membrane-anchored AAA+ (<u>A</u>TPases <u>A</u>ssociated with diverse cellular <u>A</u>ctivities) family protease and plays a pivotal role in mitochondrial proteostasis by selectively degrading misfolded and native proteins. The precise mechanisms by which nucleotide binding and hydrolysis influence YME1L's conformational dynamics, proteolytic activity, and stability remain unclear. Here, we characterize the conformational dynamics 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 Zn²⁺ binding stabilizes the protease domain. We also reveal long-range functional crosstalk between the AAA+ and protease domains of YME1L. We use functional assays to show the importance of a salt bridge between the AAA+ and protease domains 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- and heat-induced aggregation. These findings explain the nucleotide-driven regulation of YME1L and provide insights into our understanding of its proteolytic activity and structural stability under stress conditions.