鎂離子對於隆蛋白酶組裝及催化活性的結構與功能分析

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

蘇士傑(Shih-Chieh Su)

指導教授 ： 張崇毅

國立臺灣大學/生命科學院/生化科學研究所/博士(2016年)

https://doi.org/10.6342/NTU201700896

摘要

隆蛋白酶（Lon protease)是屬於ＡＴＰ依賴性的蛋白質分解酶，在生物演化中是高度保留可以存在於原核細胞內及真核生物胞器中，例如人類的粒線體及植物的葉綠體。在生物體中溶蛋白酶將細胞內蛋白質降解提供許多重要的功能，舉例來說在細胞內選擇性降解調控蛋白質使其細胞內在不同時期有不同濃度進而有效率調控，例如降解轉錄因子或者細胞週期的調控蛋白質。另外還有個重要功能是清除細胞內不正常蛋白質像是摺疊錯誤的蛋白質或是已受損的蛋白質。文獻指出隆蛋白酶需要鎂離子才可組裝成六聚體而具有功能需要有ＡＴＰ才可以降解具有二級結構或無結構的蛋白質受質。而也發現在沒有ＡＴＰ只有鎂離子情況下隆蛋白酶可以降解無二級結構的受質。目前沒有研究解釋這現象。
利用低溫電子顯微鏡(Cryo-EM)建構隆蛋白酶在有鎂離子情況下的蛋白質結構。發現鎂離子會讓隆蛋白酶形成六聚體但是側邊有縫隙並不像有ＡＴＰ情況下形成封閉的六聚體。而建構出的蛋白質結構可以解釋沒有二級結構的蛋白質受質可以藉由擴散的方式進入到隆蛋白酶的催化活性的位置進而將受質分解而且不需依賴ＡＴＰ。也利用結晶學發現鎂離子結合到隆蛋白酶的催化活性位置附近進而改變 substrate-binding loop促進隆蛋白酶的降解活性。以上的結果指出鎂離子對於隆蛋白酶的組裝及活性是扮演重要的角色。

關鍵字

隆蛋白酶 ； 低溫電子顯微鏡

並列摘要

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.

並列關鍵字

Lon protease ； Cryo-EM

參考文獻

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