Capturing oligomeric states of Lon protease involved in its regulation using CryoEM

Authors

• I Rathore
• A Gustchina
• D Zhang
• A Andrianova
• A Kudzhaev
• I Smirnov
• T Rotanova
• A Wlodawer

Abstract

Lon is an AAA+ serine protease crucial for protein quality control in eukaryotes and prokaryotes. Dysregulation of Lon leads to diseases like mitochondrial encephalomyopathy, cancer, and CODAS syndrome. Despite its ubiquity, the regulation of Lon protease activity remains unclear. Functionally active as a hexamer, Lon has been suggested to form a dodecameric state acting on peptide substrates. We hypothesize that dodecamer formation is a regulatory state of Lon and aim to elucidate its structural basis.
We report the formation of higher-order oligomers of E. coli Lon (EcLon) and identify different states using CryoEM. Utilizing Mass Photometry shows the formation of dodecamer (12mer), undecamer (11mer), and decamer (10mer) states. Seven nanobodies were developed to stabilize these states for structural elucidation. High-resolution structures of the 12mer, 11mer, and 10mer oligomeric states were determined, formed by 6+6mer, 6+5mer, and 5+5mer respectively. The interface between these arrangements involves the intricate interaction of Lon N-terminal domain (NTD). The NTD interface arrangement differs among the three configurations. The 12mer and 11mer states were identified in the apo form, while the NTD-binding nanobody enriched the 10mer state in CryoEM studies. The 12mer forms a symmetrical arrangement with a contiguous channel connecting hexamers, whereas the 11mer and 10mer have oligomers arranged at an angle, with the NTD blocking the contiguous channel formation. Based on CryoEM structures, we hypothesize that Lon's different states regulate its activity and the number of active proteins. We aim to trap different EcLon states with nanobodies and conduct experiments to assign their functional relevance.

Scientific Focus Area: Structural Biology

This page was last updated on Tuesday, August 6, 2024