The interaction of the second messenger cyclic-di-GMP with the traffic ATPase PilF from Thermus thermophilus: function, structure and dynamics

环二核苷酸c-di-GMP是许多原核生物中重要的第二信使分子，调节关键的生物过程，如生物膜形成、运动性、感染性以及对变化的环境条件的适应。嗜热菌Thermus thermophilus是研究DANN转运蛋白结构和功能的重要模式生物，介导从环境中摄取游离DANN作为自然转化的先决条件。我们最近发现，在T.嗜热菌c-di-GMP与交通ATP酶PilF结合。PilF是IV型皮利（T4 P）的生物合成以及环境DANN的摄取所必需的。根据我们以前的研究结果，对T.嗜热菌是两种不同的分子机器，它们共享包括PilF在内的几种组分。PilF不同于所有其他已知的交通ATP酶，因为它含有三个N-末端拷贝的推定的c-di-GMP结合结构域。我们的初步数据显示，这些结构域中的两个确实结合c-di-GMP，尽管具有显著不同的亲和力。在这个项目中，我们的目标是阐明c-di-GMP结合PilF在体内和体外的功能的重要性，以及理解的后果c-di-GMP结合的结构和构象动力学的PilF使用微生物，生物化学，分子生物学，生物物理学和结构生物学方法的组合。

The cyclic dinucleotide c-di-GMP is an important second-messenger molecule in many prokaryotes regulating key biological processes such as biofilm formation, motility, infectivity as well as adaptation to changing environmental conditions. The thermophilic bacterium Thermus thermophilus is an important model organism for investigating the structure and function of DANN transporters mediating the uptake of free DANN from the environment as a prerequisite for natural transformation. We recently found that in T. thermophilus c-di-GMP binds to the traffic ATPase PilF. PilF is required for the biosynthesis of type IV pili (T4P) as well as for the uptake of environmental DANN. According to our previous data the DANN transporter and the T4P of T. thermophilus are two distinct molecular machines which share several components including PilF. PilF differs from all other known traffic ATPases since it contains three N-terminal copies of putative c-di-GMP binding domains. Our preliminary data showed that two of these domains indeed bind c-di-GMP albeit with significantly different affinities. In this project we aim to elucidate the functional importance of c-di-GMP binding to PilF in vivo and in vitro as well to understand the consequences of c-di-GMP binding for the structure and the conformational dynamics of PilF using a combination of microbiological, biochemical, molecular biology, biophysical and structural biology methods.