La蛋白酶N端功能區對於受質辨識所扮演的角色

Abstract

Protease la又稱為Lon AAA+蛋白酶 (LonA) 是第一個被發現的ATP依賴型蛋白酶，屬於多功能性的蛋白，在進行生物功能時會由單一種單體聚合成多聚體，在古細菌、原核生物及真核生物的胞器中都具有LonA。LonA蛋白酶在生物體中主要藉由分解不正常或是損壞的蛋白進行蛋白質的品質管理，避免廢物在細胞中累積，除此之外，LonA也參與多種生物功能的調控。先前在台灣本土嗜熱菌Meiothermus taiwanensis LonA蛋白酶 (MtaLonA) 的研究中發現，N端功能區與因為溫度影響而造成結構部分展開的受質 (Ig2) 之降解相關，然而對於LonA N端功能區到底如何辨識受質並與受質結合詳細的機制仍不清楚，因此本研究想利用核磁共振實驗以結構生物學的角度探討LonA N端功能區與受質間的交互用關係，藉此了解N端功能區所扮演的角色。在全長的MtaLonA異核核磁共振實驗中，我們得到一個有約兩百個清楚交叉峰的光譜，而這些訊號大致上與MtaLonA N端截短區塊 (NN206) 的交叉峰重疊，顯示MtaLonA的N端功能區以一個較為動態的接鏈和ATPase功能區及蛋白酶功能區連接，使得N端功能區能夠獨立的在環境中擺動，因此我們也得以利用核磁共振實驗觀察N端功能區的化學位移擾動，除此之外，利用多維異核核磁共振實驗，我們完成了NN206約95%的骨架循序判定。將受質加入NN206中並加熱使受質結構逐漸展開後，從核磁共振光譜中可以觀察到NN206的化學位移發生劇烈的改變，而根據NN206的結構及循序判定的結果也發現受到影響的殘基主要位於N端子功能區，我們也進一步對NN206進行點突變，並且發現NN206 N端子功能區暴露於外界環境中的疏水性殘基對於與未折疊的受質結合是必須的。綜合上述結果，本篇實驗發現MtaLonA藉由N端功能區中N端子功能區的疏水性殘基辨識未折疊或聚集的蛋白。

Lon AAA+ protease (LonA), the first ATP-dependent protease to be identified, is a multi-functional, homo-oligomeric enzyme. LonA plays a crucial role in protein quality control and regulation of diverse biological processes in prokaryotes and eukaryotic organelles. Previous studies suggest that N-terminal domain (NTD) of Meiothermus taiwanensis LonA (MtaLonA) is required for a thermally unfolded tandem immunoglobulin substrate from Dictyostelium discoideum (Ig2) binding. However, the structural basis for the substrate recognition mediated by the NTD domain has not been characterized. Here we seek to understand how the NTD of LonA recognizes and traps protein substrates by Nuclear Magnetic Resonance (NMR) spectroscopy. Heteronuclear NMR analyses show 200 well-resolved 15N-1H correlations of full-length hexameric MtaLonA at an apparent molecular weight of 0.5 MDa. Importantly, most of these correlations are superimposable to those observed in the N-terminal truncated form (NN206) spectrum. The results reveal that the NTD is loosely linked to the hexameric core via a flexible linker rendering it possible for detailed chemical shift perturbation mapping of substrate binding in the context of full-length MtaLonA. We also apply multidimensional heteronuclear NMR experiments to assign 95% of the non-proline backbone 1H and 15N resonances of NN206. Moreover, the chemical shifts of NN206 are significantly perturbed by the addition of damaged substrate and structural mapping of the chemical shift perturbations shows that the affected residues are mainly located in the N subdomain. We further show exposed hydrophobic residues are required for interaction with unfolding conformation by mutagenesis. Together, our findings demonstrate that MtaLonA recognizes unfolded or aggregated substrate via exposed hydrophobic residues located in the N subdomain of its NTD.