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標題: Xanthomonas campestris之GGDEF蛋白XCC4471與c-di-GMP複合體的結構解析與diguanylate cyclase活性分析
The structure and inhibition of a GGDEF diguanylate cyclase complexed with (c-di-GMP)2 at the active site
作者: 楊超宇
Yang, Chao-Yu
關鍵字: Xanthomonas campestris;十字花科黑腐病菌;diguanylate cyclase;c-di-GMP;鳥甘酸環化酶;環狀二鳥甘酸
出版社: 生物化學研究所
c-di-GMP是細菌中一個重要的訊息傳遞分子,參與調控細胞中許多重要的生物功能,包括毒性因子的表達、生物膜的合成以及細胞的運動…等。c-di-GMP的合成主要是由具有GGDEF domains的diguanylate cyclase(DGC)所催化合成的,而它的活性是藉著與其產物(c-di-GMP)結合於其異位抑制區(allosteric inhibitory (I) site)來調控。然而卻有一大部分的GGDEF domains欠缺了與c-di-GMP結合的異位調控區之高保留序列RxxD。在本研究中,我們利用X-ray的晶體繞射技術解析了Xanthomonas campestris DGC之GGDEF domain XCC4471GGDEF與c-di-GMP之複合體結構。觀察此複合體之三度空間結構,我們驚訝地發現兩分子XCC4471GGDEF藉著與兩分子c-di-GMP結合於高保留活性區而相互連結形成二聚體。在此複合體結構中,(c-di-GMP)2採取一種前所未見的部分地相互穿插之形式,座落於兩個面對面的XCC4471GGDEF中央,其外圍兩個guanine bases結合於guanine-binding pockets,而中心兩個guanine bases則相互堆疊。利用單點突變的方式,改變XCC4471GGDEF中參與c-di-GMP專一結合的胺基酸,都會導致其與c-di-GMP結合能力顯著下降。另外,至今數以萬計的GGDEF domains中,這些胺基酸都是高度保留的。以上這些結果顯示了GGDEF蛋白結合(c-di-GMP)2於活性區的一個新的product-bound模式。此嶄新的XCC4471GGDEF-c-di-GMP複合體結構或許可以提供一個普遍模型,幫助我們針對X. campestris或者是其它也具有多種GGDEF蛋白的微生物,設計能夠阻斷DGC活性之藥物前驅物。

c-di-GMP is a key signalling molecule involved in regulating many important biological functions in bacteria. The synthesis of c-di-GMP is catalyzed by the GGDEF-domain-containing diguanylate cyclase (DGC), the activity of which is regulated by the binding of product at the allosteric inhibitory (I) site. However, a significant number of GGDEF domains lack the RxxD motif characteristic of the allosteric I site. Here, the structure of XCC4471GGDEF, the GGDEF domain of a DGC from Xanthomonas campestris, in complex with c-di-GMP has been solved. Unexpectedly, the structure of the complex revealed a GGDEF-domain dimer cross-linked by two molecules of c-di-GMP at the strongly conserved active sites. In the complex (c-di-GMP)2 adopts a novel partially intercalated form, with the peripheral guanine bases bound to the guanine-binding pockets and the two central bases stacked upon each other. Alteration of the residues involved in specific binding to c-di-GMP led to dramatically reduced KD values between XCC4471GGDEF and c-di-GMP. In addition, these key residues are strongly conserved among the many thousands of GGDEF-domain sequences identified to date. These results indicate a new product-bound form for GGDEF-domain containing proteins obtained via (c-di-GMP)2 binding at the active site. This novel XCC4471GGDEF-c-di-GMP complex structure may serve as a general model for the design of lead compounds to block the DGC activity of GGDEF-domain containing proteins in X. campestris or other microorganisms that contain multiple GGDEF-domain proteins.
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