臺灣中度嗜熱菌MtaLonC蛋白酶N端區域之晶體結構分析

Structural analysis of the N-terminal domain of MtaLonC from Meiothermus taiwanensis

黎振凱(Jhen-Kai Li)

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/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10496

摘要

Lon 蛋白脢是一種需要ATP來進行其活性的酵素，Lon 蛋白脢屬於AAA+
超級家族的成員之一，這些Lon 蛋白脢主要分成兩種群體， Lon A 以及 Lon B，然而在最近的研究中指出，一種新的Lon 蛋白脢被鑑定出來，被稱為Lon C蛋白脢，這是一種不需要ATP就可以執行其活性的蛋白脢酵素，我們的實驗團隊從一
種中度嗜熱菌分離出LonC蛋白脢，稱為MtaLonC， 並且將分離出的MtaLonC
以基因工程的方法重新純化出重組蛋白，並且將MtaLonC的結構所解出來，
MtaLonC形成一種類似桶狀的結構，並且開口與出口相通形成中空相連的結構，
由此結構可以指出MtaLonC的N-端區域與一種類似AAA+的結構相連接，而
MtaLonC的N-端區域的序列與其它種Lon蛋白脢分析過後發現無相似之處，而
在此全長結構的N-端區域缺少一部分的電子密度圖，並推測結構可能具有可變
性•為了將缺少電子密度圖的N-端結構解出以及了解其功能，MtaLonC的N-端
區域的重組蛋白被建構出來並且將其蛋白進行結晶，以進行X光散射實驗，
MtaLonC的N-端區域晶體的散射數據達到2.4 A，並且成功的利用多波長散射來
解出結構，此解出的結構呈現一個巨大的ɑ-螺旋髮夾結構(HHE)從AAA+相似結
構所突出•研究結果顯示這種可變性的HHE結構與不完整結構蛋白的結合以及辨
識有關，並且防止不完整結構蛋白互相聚集，因此MtaLonC不只是一種降解機器
也扮演著蛋白質監控者的角色•

關鍵字

MtaLonC ； 晶體結構 ； HHE

並列摘要

Lon protease is an ATP-dependent protease belonging to the AAA+ (ATPases associated with diverse cellular activities) superfamily of enzymes. These Lon proteases have been classified into two groups, Lon-A and Lon-B. Recently, Lon-C, a novel family of Lon-like proteases with no ATPase activity has been identified and the crystal structure of MtaLonC from a thermophilic bacterium determined by our lab. MtaLonC forms a barrel-like structure with open axial pores. The structure showed that the N-terminal domain of MtaLonC adopts an AAA-like fold, which was not detected by previous sequence analysis. A significant portion of the N-terminal domain is disordered in the structure of the full-length MtaLonC. To obtain insights into the structure and function of the N-terminal domain, an N-terminal construct of MtaLonC was crystallized, and X-ray diffraction data was collected to 2.4 A. The crystal structure was successful determined by multiple anomalous dispersions (MAD). The crystal structure showed that a large ɑ-helical hairpin extension (HHE) protrudes from the AAA-like domain. We also showed that the flexible HHE may be involved in recognition/binding of unfolded protein substrates prior to their entry through the pore. These results suggest that MtaLonC is a degradation machine with chaperone activity.

並列關鍵字

MtaLonC ； crystal structure ； HHE

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