DIMERIZATION AND ACTIVATION MECHANISM OF PKNB, A SERINE/THREONINE KINASE FROM MT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 结核分枝杆菌(MTB)含有11个预测的真核生物样丝氨酸/苏氨酸蛋白激酶(STPKs)。PKnB是一种MTB STPK，它介导的信号转导调节细胞的形状，可能还调节细胞的分裂。在这项研究中，我们将检查高度保守的PounB二聚界面的意义，它位于活性中心的对面。对一个二聚界面突变体(L33D)的初步结构测定显示，一个单体缺少关键的催化接触，质谱分析表明，与野生型PnuB相比，磷酸化程度较低。此外，初步的生化数据表明，二聚体的形成促进了PnuB的自动磷酸化，并且这一过程是分子间的。为了进一步研究PuncB的激活机制，我们有兴趣单独使用催化死亡突变体(D138N)，并结合二聚体界面突变来确定失活蛋白激酶的晶体结构。我们也希望获得更高质量的L33D结构的原子分辨率数据。这些研究将进一步确定STPKs的调节机制，并有助于开发新的结核分枝杆菌疗法。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mycobacterium tuberculosis (MTB) contains 11 predicted eukaryotic-like serine/threonine protein kinases (STPKs). Signal transduction mediated by PknB, an MTB STPK, regulates cell shape, and possibly cell division. In this study, we will examine the significance of the highly conserved PknB dimerization interface, which is located on the side opposite the active site. Initial structural determination of a dimerization interface mutant (L33D) showed a monomer which was missing critical catalytic contacts, and mass spectrometry indicated less phosphorylation than wild-type PknB. Additionally, preliminary biochemical data suggests that PknB autophosphorylation is enhanced by dimer formation and that this process is intermolecular. To further characterize the activation mechanism of PknB, we are interested in determining the crystal structures of the inactive kinase using a catalytically dead mutant (D138N) alone, and in combination with the dimer interface mutation. We also hope to obtain higher-quality atomic resolution data for the L33D structures. These studies will further define the regulatory mechanisms of STPKs and aid in the development of novel MTB therapeutics.