Mechanism of Urease Metallocenter Biosynthesis

描述(由申请人提供)：本提案中描述的研究集中在细菌毒力因子细菌尿素酶的激活机制上。尿素酶脱脂蛋白(UreABC)是一种新型的赖氨酸氨基甲酸酯桥联的双核镍活性中心金属分配中心的骨架。我们的组装过程工作模型需要依赖GTP的分子伴侣(由Our、UreF和UreG辅助蛋白组成)的作用，以及提供镍的金属配位伴侣(UreE)的参与。我们试图了解每个尿素酶辅助蛋白的结构和功能，并阐明这些蛋白参与这一独特的依赖GTP的金属分配中心组装过程的机制。我们的目标是测试关于尿素酶辅助蛋白作用的特定假说。因此，我们将(A)进行单个ORED、UreF和UreG组分以及UreDFG杂三聚体的结构分析。(B)揭示与Our-UreABC、UreDF-UreABC和UreDFG-UreABC物种形成相关的顺序结合动力学。(C)用小角X射线散射法确定这些络合物的整体构象。(D)确定蛋白质的具体位置：通过开发一种创新的“蛋白质足迹”技术确定蛋白质相互作用，该技术应广泛适用于其他系统。(E)通过比较有无被重氮甲烷捕获的放射性标记二氧化碳的掺入情况，探索这些复合体中赖氨酸氨甲酰化反应的时机和稳定性。(F)寻求通过使用突变体和交换惰性金属离子来稳定假定的UreE：UreG相互作用。(G)使用等温滴定量热法和其他金属结合方法，测试在UreE：UreG界面上创建高亲和力镍结合位点的情况。(H)研究镁GTP及其类似物对金属转移到UreABC的构象变化的影响。尿素酶金属分配中心生物合成的一般过程可能也适用于许多其他系统，包括许多医学上有价值的金属酶的激活。值得注意的是，我们对了解尿素酶激活的关注将揭示可以应用于这些不太容易处理的系统的共同原则。此外，我们还将扩大我们对镍的生物化学的了解。

DESCRIPTION (provided by applicant): The studies described in this proposal focus on the mechanism of activation of bacterial urease, a bacterial virulence factor. The urease apoprotein (UreABC) serves as the scaffold for creation of a novel lysine carbamate-bridged, dinuclear nickel active site metallocenter. Our working model of the assembly process requires the action of a GTP-dependent molecular chaperone (made up of UreD, UreF, and UreG accessory proteins) along with the participation of a metallochaperone (UreE) that delivers Ni. We seek to understand the structure and function of each urease accessory protein and to elucidate the mechanism by which these proteins participate in this unique GTP-dependent process of metallocenter assembly. Our objectives are to test specific hypotheses regarding the roles of the urease accessory proteins. Thus, we will (A) Carry out structural analyses of the individual UreD, UreF, and UreG components along with the UreDFG heterotrimer. (B) Unravel the sequential binding kinetics associated with formation of the UreD-UreABC, UreDF-UreABC, and UreDFG-UreABC species. (C) Establish the overall conformation of these complexes by small angle X-ray scattering methods. (D) Identify the specific sites of protein:protein interaction by developing an innovative "protein footprinting" technology that should be widely applicable to other systems. (E) Explore the timing and stability of lysine carbamylation within these complexes by comparing the incorporation of radiolabeled CO2 with and without trapping by diazomethane. (F) Seek to stabilize the putative UreE:UreG interaction by use of mutants and exchange-inert metal ions. (G) Test for the creation of a high affinity Ni-binding site at the UreE:UreG interface by using isothermal titration calorimetry and other metal-binding approaches. (H) Examine the effect of MgGTP and its analogues on conformational changes associated with metal transfer into UreABC. The general processes involved in urease metallocenter biosynthesis are likely to apply to numerous other systems, including the activation of many metalloenzymes of medical interest. Significantly, our focus on understanding urease activation will uncover common principles that can be applied to these less tractable systems. In addition, we will expand our knowledge regarding the biochemistry of nickel.