2012年3月11日,，清華大學(xué)生命學(xué)院施一公教授研究組在《自然》（Nature）在線發(fā)表了名為“Structure and mechanism of a glutamate-GABA antiporter”的科研論文,，報道了大腸桿菌谷氨酸：γ-氨基丁酸（GABA）反向轉(zhuǎn)運蛋白(GadC)的晶體結(jié)構(gòu),，并結(jié)合生化實驗提出了GadC轉(zhuǎn)運底物的可能機制,。
2011年在歐洲爆發(fā)的腸溶血性大腸桿菌疫情導(dǎo)致數(shù)千人感染,，多人死亡,，并引起了極大的社會恐慌。大腸桿菌O104:H4菌株是這一疫情的罪魁禍首,。食物來源的大腸桿菌必須通過極酸的胃環(huán)境(pH~2)才能到達腸道，為了保證在如此低的pH下仍然能夠存活，大腸桿菌進化出了多個抗酸系統(tǒng)來對抗極酸環(huán)境,。因此研究大腸桿菌抗酸機制對人類健康有直接的重要意義,。
大腸桿菌抗酸系統(tǒng)II(Acid Resistance system 2)通過谷氨酸:γ-氨基丁酸反向轉(zhuǎn)運蛋白(GadC)將細胞外的谷氨酸轉(zhuǎn)運到細胞內(nèi),，在胞漿內(nèi)谷氨酸發(fā)生脫羧反應(yīng)，消耗一個質(zhì)子生成γ-氨基丁酸,，而產(chǎn)物γ-氨基丁酸再被GadC轉(zhuǎn)運到細胞外,。整個過程相當于向細胞外排出一個質(zhì)子，降低了細胞內(nèi)的質(zhì)子濃度,，從而起到了抗酸的作用,。理解GadC的工作機理對于研究腸道致病菌抗酸系統(tǒng)十分關(guān)鍵。從GadC的三維空間結(jié)構(gòu)信息入手,，施一公教授研究組解析了大腸桿菌GadC高分辨率的晶體結(jié)構(gòu),。結(jié)構(gòu)分析表明,，含有12個跨膜螺旋的GadC在pH堿性條件下呈現(xiàn)出轉(zhuǎn)運通道開口朝向胞內(nèi)的構(gòu)象,，令人驚奇的是,，GadC羧基端結(jié)構(gòu)域在細胞內(nèi)一側(cè)像塞子一樣將轉(zhuǎn)運通道封閉住。同時進一步的生化實驗表明，GadC對底物的轉(zhuǎn)運嚴格依賴于環(huán)境pH值：野生型GadC在pH小于6.5的環(huán)境下才具有轉(zhuǎn)運能力,，而在pH大于6.5的環(huán)境中完全沒有活性。羧基端“塞子”結(jié)構(gòu)域在GadC感受pH的過程中起到了重要的調(diào)節(jié)作用。這樣一種機制既保證了抗酸系統(tǒng)在極酸環(huán)境中能夠啟動并轉(zhuǎn)運底物,，又防止其在正常生理條件下造成胞內(nèi)質(zhì)子不必要的外流。
以上研究成果為進一步研究大腸桿菌抗酸機制提供了重要線索,。這是施一公教授研究組繼2009年、2010年報道Arginine:Agmatine反向轉(zhuǎn)運蛋白AdiC的結(jié)構(gòu)與機理之后,，在腸道細菌抗酸性研究中的又一重要成果,。（生物谷 bioon.com）
doi:10.1038/nature10917
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Structure and mechanism of a glutamate-GABA antiporter
Dan Ma, Peilong Lu, Chuangye Yan, Chao Fan, Ping Yin, Jiawei Wang，Yigong Shi
Food-borne hemorrhagic Escherichia coli, exemplified by the strains O157:H7 and O104:H4, require elaborate acid-resistance systems (ARs)to survive the extremely acidic environment such as the stomach (pH≈2). AR2 expels intracellular protons through the decarboxylation of l-glutamate (Glu) in the cytoplasm and exchange of the reaction product γ-aminobutyric acid (GABA) with extracellular Glu. The latter process is mediated by the Glu–GABA antiporter GadC, a representative member of the amino-acid–polyamine-organocation superfamily of membrane transporters. The functional mechanism of GadC remains largely unknown. Here we show, with the use of an in vitro proteoliposome-based assay, that GadC transports GABA/Glu only under acidic conditions, with no detectable activity at pH values higher than 6.5. We determined the crystal structure of E.coli GadC at 3.1 A resolution under basic conditions. GadC, comprising 12 transmembrane segments (TMs), exists in a closed state, with its carboxy-terminal domain serving as a plug to block an otherwise inward-open conformation. Structural and biochemical analyses reveal the essential transport residues, identify the transport path and suggest a conserved transport mechanism involving the rigid-body rotation of a helical bundle for GadC and other amino acid antiporters.
