Functions and mechanisms of M. tuberculosis S/T kinases

描述（由申请人提供）：本计划的长期目标是了解最近在结核分枝杆菌（Mtb）中发现的Ser/Thr蛋白激酶（STPKs）的功能。这种致病细菌是结核病（TB）的病原体，感染了世界三分之一的人口。结核病每年造成300多万人死亡，比任何其他传染病都多。尽管对蛋白激酶和磷酸酶进行了大量的研究，但对Mtb STPK和单个Mtb Ser/Thr磷酸酶Ppp的作用知之甚少。需要研究确定这些酶的特定生物学功能，以了解环境信号如何调节结核分枝杆菌代谢。 我们将集中生物化学，生物物理，结构和遗传学方法主要对四个结核分枝杆菌蛋白。这项研究有四个具体目标： 1.定义两种Mtb跨膜受体激酶中信号传导的基础。 2.定义PknB激酶在关闭和打开状态之间切换的机制。 3.发现结核分枝杆菌STPKs的蛋白质底物。 4.定义细胞内信号通路，重点是单一的Mtb Ser/Thr磷酸酶，Ppp。 我们的初步研究确定了这些目标的可行性。我们对Mtb PknB激酶结构域的生物化学和结构研究表明，原核和真核STPKs具有非常保守的三维结构和普遍的调节和底物识别机制。我们确定了细菌STPK传感器结构域和细菌Ser/Thr磷酸酶的第一晶体结构。我们已经为定义PknB激酶的活性和非活性状态做好了准备。我们对PknB和Ppp底物的探索将开始定义这些蛋白质的功能。由于激酶信号在细胞生理学中的核心作用、STPK作为药物靶点的日益关注以及结核病对全球健康的影响，我们提出的研究将对分子生物学和医学具有重要意义。

DESCRIPTION (provided by applicant): The long-term goal of this proposed program is to understand the functions of Ser/Thr protein kinases (STPKs) recently discovered in Mycobacterium tuberculosis (Mtb). This pathogenic bacterium, which is the causative agent of tuberculosis (TB), infects one third of the world's population. TB kills over 3 million people annually, more than any other infectious disease. Despite the large amount of work on protein kinases and phosphatases, little is known about the roles of the Mtb STPKs and the single Mtb Ser/Thr phosphatase, Ppp. Research to define the specific biological functions of these enzymes is needed to know how environmental signals regulate Mtb metabolism. We will focus biochemical, biophysical, structural and genetic methods principally on four Mtb proteins. This research has four specific aims: 1. Define the basis for signaling in two Mtb transmembrane receptor kinases. 2. Define the mechanism of PknB kinase switching between off and on states. 3. Discover protein substrates of Mtb STPKs. 4. Define the intracellular signaling pathways, focusing on the single Mtb Ser/Thr phosphatase, Ppp. Our preliminary studies established the feasibility of these aims. Our biochemical and structural work on the kinase domain of Mtb PknB showed that the prokaryotic and eukaryotic STPKs share a remarkably conserved three-dimensional structure and universal mechanisms of regulation and substrate recognition. We determined the first crystal structures of a bacterial STPK sensor domain and bacterial Ser/Thr phosphatase. We have set the stage to define the active and inactive states of the PknB kinase. Our proposed exploration of the substrates of PknB and Ppp will begin to define the functions of these proteins. Because of the central roles of kinase signaling in cellular physiology, the increasing focus on STPKs as pharmaceutical targets, and the worldwide health impact of TB, our proposed studies will have high significance for molecular biology and medicine.