Structural Studies of the MPT64 Family of Mycobacterial Virulence Factors

描述(申请人提供)：MPT64蛋白及其同源物形成了在致病分支杆菌属中发现的高度保守的分泌蛋白家族。该家族的创始成员来自结核分枝杆菌(MPT64，蛋白Rv1980c)，只有在分枝杆菌细胞活跃分裂时才表达。凭借这一相对独特的表达谱，Rv1980c目前正在南非和秘鲁进行第三阶段临床试验，以评估其取代结核菌素或纯化蛋白衍生物作为活动性结核病感染基准诊断的潜力。尽管NIH赞助了许多支持结核病研究的活动，并且Rv1980c对于改进世界上最广泛的单一细菌传染病的诊断具有潜在的重要性，但传统的遗传和生化方法未能就Rv1980c或该蛋白质家族的任何其他成员的生物学作用提供任何解释(S)。为了满足这一需求，我们建议使用结构生物学中最先进的方法，通过以下特定目标更好地了解MPT64家族的生物学作用：特定目标1：通过异核核磁共振光谱确定Rv1980c的高分辨率溶液结构集合。具体目的2：通过解析鸟分枝杆菌亚种基因组中发现的2个家族成员中的1个MAP0087的高分辨晶体结构，研究MPT64家族内部结构上的相似性和差异性。副结核(地图)。具体目的3：利用异核核磁共振技术比较野生型、二硫键连接的Rv1980c和非连接的突变蛋白的结构和动力学性质，为深入了解Rv1980c的皮肤屏障转运机制提供依据。AIMS 1和AIMS 2的结果将通过与已知功能蛋白质的结构同源性，将可能的生物学角色关联到MPT64家族成员，这将进一步加深我们对这些蛋白质在结核分枝杆菌和相关生物复杂发病机制中的理解。AIM 3的结果将提供对Rv1980c皮肤屏障转运的机械洞察，并可能建议改进这一有前途的诊断试剂的方法。这里提出的研究有可能对广泛传播的分枝杆菌疾病的治疗和诊断产生影响，因此属于该机构的任务。

DESCRIPTION (provided by applicant): The MPT64 protein and its homologs form a highly conserved family of secreted proteins found within the pathogenic Mycobacteria genus. The founding member of this family from Mycobacterium tuberculosis (MPT64, protein Rv1980c) is expressed only when the Mycobacteria cells are actively dividing. By virtue of this relatively unique expression profile, Rv1980c is currently under phase III clinical trials in South Africa and Peru to evaluate its potential to replace tuberculin, or purified protein derivative, as a benchmark diagnostic for active Tuberculosis infection. Despite numerous NIH-sponsored initiatives supporting Tuberculosis research and the potential importance of Rv1980c to the improved diagnosis of the single most widespread bacterial infectious disease in the world, traditional genetic and biochemical approaches have failed to provide any explanation as to the biological role(s) of Rv1980c or any other members of this family of proteins. To address this need, we propose the use of state of the art methods in structural biology to provide a better understanding of the biological roles of the MPT64 family through the following specific aims: Specific Aim 1: To determine an ensemble of high-resolution solution structures for Rv1980c by heteronuclear NMR spectroscopy. Specific Aim 2: To investigate the areas of structural similarity and divergence within the MPT64 family by solving the high-resolution crystal structure of MAP0087, 1of 2 family members found in the genome of Mycobacterium avium subsp. paratuberculosis (Map). Specific Aim 3: To use heteronuclear NMR to compare the structural and dynamic properties of wild type, disulfide-linked Rv1980c with a non-linked mutant protein to provide insights into the mechanism of Rv1980c skin barrier transit. The results of Aims 1 and 2 will be correlated and used assign likely biological roles to MPT64 family members by virtue of structural homology to proteins of known function; this will further our understanding of these proteins in the complex pathogenesis of M. tuberculosis and related organisms. The results of Aim 3 will provide mechanistic insight into Rv1980c skin barrier transit and may suggest approaches to improving this promising diagnostic agent. The studies proposed here have the potential to impact both the treatment and diagnosis of widespread Mycobacterial disease, and therefore lie within the mission of the agency.