上海藥物所抗菌交叉團(tuán)隊(duì)研究人員針對金黃色葡萄球蛋白水解酶ClpP（SaClpP）,，開展水解功能的分子機(jī)制研究，繼2011年在美國生物化學(xué)和分子生物學(xué)學(xué)會專業(yè)期刊《Journal of Biological Chemistry》上發(fā)表論文之后,，研究論文再次發(fā)表于該雜志（J Biol Chem,，2013,，288，17643–53）,。
ClpP是細(xì)菌中關(guān)鍵的蛋白水解系統(tǒng),，由上下兩個七聚體自組裝成一個球形腔體，負(fù)責(zé)維持細(xì)菌體內(nèi)蛋白質(zhì)的穩(wěn)態(tài),，對維系細(xì)菌的致病力起著關(guān)鍵作用?，F(xiàn)有研究表明ClpP已成為一類針對新機(jī)制的抗菌新靶標(biāo)，目前還沒有靶向調(diào)控劑處于臨床研究,。繼該研究團(tuán)隊(duì)獲得的SaClpP的兩種功能構(gòu)象：Extended和Compressed構(gòu)象的基礎(chǔ)上,，羅成與楊財廣研究員指導(dǎo)博士生葉飛和張婕，以長時間分子動力學(xué)模擬為導(dǎo)向,，結(jié)合生化實(shí)驗(yàn),，闡明了SaClpP構(gòu)象變化的具體路徑和驅(qū)動力；利用分子動力學(xué)捕捉到了SaClpP在兩種構(gòu)象間轉(zhuǎn)化的中間態(tài)Compact構(gòu)型,，通過晶體結(jié)構(gòu)的解析肯定了該中間構(gòu)象的存在和合理性,；再通過理論模擬發(fā)現(xiàn)了構(gòu)象變化過程中的數(shù)個重要位點(diǎn)（如A140、E137等）,，得到了實(shí)驗(yàn)驗(yàn)證,。
最終，本研究通過理論模擬與實(shí)驗(yàn)驗(yàn)證緊密結(jié)合,，闡明了SaClpP的動態(tài)調(diào)控機(jī)制：多肽底物進(jìn)入ClpP空腔后被催化位點(diǎn)降解,，降解產(chǎn)物在活性中心的積累造成局部分子間相互作用方式的改變，引起活性位點(diǎn)殘基構(gòu)象的變化,，進(jìn)而傳遞到附近的R171_D170網(wǎng)絡(luò),，使得該網(wǎng)絡(luò)中的相互作用也遭到破壞，Extended構(gòu)型不能保持穩(wěn)定,，逐漸轉(zhuǎn)變成Compressed構(gòu)型,，該構(gòu)型被E137網(wǎng)絡(luò)所穩(wěn)定。在構(gòu)象轉(zhuǎn)化過程中,，Extended構(gòu)型中的Helix E通過解旋/再折疊的過程向Compressed構(gòu)型轉(zhuǎn)化,，A140在Helix E上扮演了一個“轉(zhuǎn)軸”的角色。
由于ClpP在不同細(xì)菌中高度保守,，該研究結(jié)果對ClpP家族其它成員的結(jié)構(gòu)及機(jī)制研究具有重要的參考意義,，也為后續(xù)開展基于調(diào)控機(jī)制研究的化學(xué)生物學(xué)和抗菌藥物發(fā)現(xiàn)研究奠定了堅(jiān)實(shí)的理論和結(jié)構(gòu)基礎(chǔ)。
該研究得到了藥物所抗菌交叉團(tuán)隊(duì),、基金委和科技部課題的資助,，該研究也獲得了中科院網(wǎng)絡(luò)中心、天津和上海超級計算中心在計算資源上的支持,。（生物谷Bioon.com）
doi:10.1074/jbc.M113.452714
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Helix Unfolding/Refolding Characterizes the Functional Dynamics of Staphylococcus aureus Clp Protease*
Fei Ye‡1, Jie Zhang‡1, Hongchuan Liu‡, Rolf Hilgenfeld‡,§2, Ruihan Zhang‡, Xiangqian Kong‡, Lianchun Li‡, Junyan Lu‡, Xinlei Zhang‡,¶, Donghai Li‖, Hualiang Jiang‡3, Cai-Guang Yang‡,**4 and Cheng Luo‡5
The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala140 acted as a “hinge” during the transition between the extended and compressed states, and Glu137 was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.
