RUB和馬普研究所的研究人員揭示了細(xì)胞轉(zhuǎn)運(yùn)調(diào)控蛋白的新作用機(jī)制，他們發(fā)現(xiàn)核心開(kāi)關(guān)蛋白R(shí)ab的活性受到其互作蛋白兩個(gè)指狀結(jié)構(gòu)的調(diào)解,，文章發(fā)表在美國(guó)國(guó)家科學(xué)院院刊PNAS雜志上,。調(diào)控細(xì)胞生長(zhǎng)的Ras蛋白只受到一個(gè)指狀結(jié)構(gòu)的調(diào)解，而Rab則需要兩個(gè)指狀結(jié)構(gòu),，研究人員由此解密了GTPases家族的新作用機(jī)制,。
GTPases是我們體內(nèi)一類(lèi)重要的小分子，Ras和Rab都是其中的一員,。Rab蛋白控制不同細(xì)胞區(qū)域之間的多種運(yùn)輸過(guò)程,，如果運(yùn)輸系統(tǒng)被破壞就會(huì)發(fā)生肥胖癥等疾病。Rab和Ras蛋白作用相似,，都類(lèi)似于一個(gè)開(kāi)關(guān),。當(dāng)開(kāi)關(guān)開(kāi)啟時(shí)高能分子GTP結(jié)合，而開(kāi)關(guān)關(guān)閉時(shí)結(jié)合的是低能分子GDP,。RabGAP蛋白負(fù)責(zé)催化GTP的水解,，在這一過(guò)程中GTP分解為GDP和一個(gè)磷酸基團(tuán)。現(xiàn)在,，研究人員以最高原子分辨率觀察并記錄了上述過(guò)程,。
動(dòng)態(tài)機(jī)制
研究人員通過(guò)X射線結(jié)構(gòu)分析，首次確定了RabGAP蛋白復(fù)合體的空間結(jié)構(gòu),。研究顯示,，這一復(fù)合體中存在兩個(gè)指狀結(jié)構(gòu)，一個(gè)精氨酸指一個(gè)谷氨酰胺指,。人們已知精氨酸指結(jié)構(gòu)也存在于Ras復(fù)合體中,，而谷氨酰胺指的存在則出人意料。研究顯示,，RabGAP的兩個(gè)指狀結(jié)構(gòu)插入Rab蛋白的GTP結(jié)合區(qū)域,，使GTP水解加速了五個(gè)數(shù)量級(jí),。
研究人員用傅里葉變換紅外光譜技術(shù)FTIR實(shí)時(shí)觀察了這一動(dòng)態(tài)過(guò)程，與X射線結(jié)構(gòu)分析相比,，傅里葉變換紅外光譜技術(shù)不僅可以提供反應(yīng)的快照還能夠捕捉到整個(gè)動(dòng)態(tài)過(guò)程,。研究顯示，正是兩個(gè)催化性指狀結(jié)構(gòu)同時(shí)插入GTP結(jié)合區(qū)域,，促使了GTP的水解,。
醫(yī)療價(jià)值
這項(xiàng)研究所用的蛋白是Rab1b和RabGAP TBC1D20，不過(guò)研究人員指出其他Rab和RabGAP與他們選取的蛋白很相似,，并由此推測(cè)其他Rab和RabGAP的活性機(jī)制也是通過(guò)兩個(gè)指狀結(jié)構(gòu)實(shí)現(xiàn)的,。GTPases家族的小分子發(fā)生突變，可能釋放出不可控的生長(zhǎng)信號(hào),，促使腫瘤形成,。研究人員認(rèn)為若能開(kāi)發(fā)出模擬雙指機(jī)制的小分子，就可以對(duì)這一過(guò)程進(jìn)行調(diào)控,，從而治療癌癥,。(生物谷Bioon.com)
doi: 10.1073/pnas.1214431110
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Catalytic mechanism of a mammalian Rab·RabGAP complex in atomic detail
Konstantin Gavriljuka,1, Emerich-Mihai Gazdagb,1, Aymelt Itzenb,2, Carsten Köttinga, Roger S. Goodyb, and Klaus Gerwerta,3
Rab GTPases, key regulators of vesicular transport, hydrolyze GTP very slowly unless assisted by Rab GTPase-activating proteins (RabGAPs). Dysfunction of RabGAPs is involved in many diseases. By combining X-ray structure analysis and time-resolved FTIR spectroscopy we reveal here the detailed molecular reaction mechanism of a complex between human Rab and RabGAP at the highest possible spatiotemporal resolution and in atomic detail. A glutamine residue of Rab proteins (cis-glutamine) that is essential for intrinsic activity is less important in the GAP-activated reaction. During generation of the RabGAP·Rab:GTP complex, there is a rapid conformational change in which the cis-glutamine is replaced by a glutamine from RabGAP (trans-glutamine); this differs from the RasGAP mechanism, where the cis-glutamine is also important for GAP catalysis. However, as in the case of Ras, a trans-arginine is also recruited to complete the active center during this conformational change. In contrast to the RasGAP mechanism, an accumulation of a state in which phosphate is bound is not observed, and bond breakage is the rate-limiting step. The movement of trans-glutamine and trans-arginine into the catalytic site and bond breakage during hydrolysis are monitored in real time. The combination of X-ray structure analysis and time-resolved FTIR spectroscopy provides detailed insight in the catalysis of human Rab GTPases
