Structural and functional analysis of the magnesium activated hexamerization and proteolytic mechanism of the Lon AAA+ protease

• Main Authors: Shih-Chieh Su, 蘇士傑
• Other Authors: Chung-I Chang
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/fydf6a

博士 === 國立臺灣大學 === 生化科學研究所 === 105 === The Lon AAA+ protease (LonA) plays critical roles in protein homeostasis and regulation of diverse biological processes. LonA is thought to form a sequestered substrate-degradation chamber by fused rings of six AAA+ and six protease domains and its activity depends on magnesium (Mg2+) and ATP. Using single-particle cryo-electron microscopy reconstructions, I show that apo-LonA without Mg2+ forms a tetrameric complex, and that binding of Mg2+ drives the formation of a hexameric chamber. Without nucleotide, both the tetrameric and hexameric LonA chambers show prominent side and top openings. The tetrameric apo-LonA is proteolytically inactive but Mg2+-activated LonA degrades efficiently unstructured protein and peptide substrates. Binding of ATP further converts the open chamber of Mg2+-activated LonA into a secluded assembly. ADP and polyatomic ions are found to inhibit proteolysis by LonA, presumably by inducing the formation of an immobile secluded chamber disallowing access of protein substrates. Moreover, to understand the role of Mg2+, I have determined a 1.85Å crystal structure of a truncated LonA bound to Mg2+ and a proteasome inhibitor, which reveals a potential Mg2+-binding site in the protease domain. Specific binding of Mg2+ is further confirmed by biophysical and biochemical assays. Structural analysis and molecular dynamics (MD) simulations suggest that binding of Mg2+ activates LonA by promoting deprotonation of the catalytic Lys required for serving as the general base, opening of the proteolytic groove, and formation of the hexameric interface of the protease ring. My findings reveal the structural roles of Mg2+, ADP, and polyatomic anions in the assembly and activity of LonA.