Bacterial back talk - unfolding and molecular interaction studies in Staphylococcal proteins and engineering structure-informed antibiotics

葡萄球菌是极为常见的人类共生菌，与医院的全身性感染、手术器械和植入物的生物膜形成、工业污染和食品腐败有关。葡萄球菌中的辅助基因调控（Agr）控制着重要的细菌功能，如毒力和表面粘附（生物膜形成）。AgrB是一种膜内肽酶，负责信号分子AIP的预处理，AIP介导细菌通讯和群体感应。AIP反应级联是一个双组分系统，包括膜受体AgrC，其磷酸化核苷酸结合伙伴AgrA。Agr蛋白的药理调控是细菌控制、减毒和生物膜抑制的一个有吸引力的靶点，也是抗菌药物设计的一个新机会。我们已经开发了一种新的分子识别试验，该试验报告了底物或配体结合通过增加蛋白质稳定性来降解。蛋白质的热展开是一个渐进的过程，其中热波动的频率和幅度随着温度的升高而增加，可以通过圆二色性、小角中子或x射线散射来监测。核磁共振波谱和高性能计算补充了Agr蛋白及其底物和调节配体之间相互作用的详细原子水平结构信息的研究。在这个项目中，我们将开发一个基于热变性实验的蛋白质/配体相互作用的大规模筛选，并将对现有的Agr调节剂文库进行验证。利用当前Agr研究项目中的重组蛋白，我们将：a)结合散射技术和CD来监测蛋白质结构和整体构象的变化，并将参考关键行为开发工具；b)在合作机构ISIS开发大型屏幕的样本环境，并结合光束线SANS2D和ZOOM；开发快速数据解释的分析框架；c)利用固态核磁共振、分子对接和模拟表征配体/受体相互作用。该计划依靠先进的生物物理、结构和计算工具进行分子分析，这些工具相互补充，但部分重叠，以确保成功完成工作。

Staphylococci are extremely common human commensals, which have been implicated in systemic infections in hospitals, biofilm formation in surgical devices and implants, industrial fouling and food spoilage. The accessory gene regulation (Agr) in staphylococci controls important bacterial functions, such as virulence and adhesion to surfaces (biofilm formation). AgrB is a membrane endopeptidase responsible for pre-processing of the signalling molecule, AIP, which mediates bacterial communications and quorum sensing. The AIP response cascade is a two component system including a membrane receptor AgrC, which phosphorylates the nucleotide-binding partner AgrA. Pharmacological regulation of the Agr protein is an attractive target for bacterial control, attenuating virulence and biofilm inhibition and a new opportunity for antimicrobial design.We have developed a new molecular recognition assay, which reports on substrate or ligand binding through increased protein stability to degradation. Thermal unfolding of proteins is a gradual process, in which thermal fluctuations increase in frequency and magnitude with temperature and can be monitored by circular dichroism, small angle neutron or X-ray scattering. NMR spectroscopy and high performance computing complement the studies with detailed atomic level structural information on the interaction between the Agr proteins and their substrates and regulatory ligands. In this project we will develop a large scale screen for protein/ligand interactions based on the thermal denaturation assay and will validate it against existing libraries of Agr modulators. Using recombinant protein from current research project on Agr, we will:a) use a combination of scattering techniques and CD to monitor changes in protein structure and overall conformation and will develop the tool with reference to critical behavior;b) develop a sample environment at partner institution ISIS for the large scale screen in conjunction with beamlines SANS2D and ZOOM; develop analytical framework for rapid data interpretation;c) characterize ligand/receptor interactions using solid state NMR, molecular docking and simulations.The programme relies on advanced biophysical, structural and computational tools for molecular analysis, which are combined in complementary, yet partially overlapping groups to ensure successful completion of the work.