Structural Studies of the MPT64 Family of Mycobacterial Virulence Factors

描述（由申请人提供）：MPT 64蛋白及其同源物形成在致病性分枝杆菌属中发现的高度保守的分泌蛋白家族。来自结核分枝杆菌的该家族的创始成员（MPT 64，蛋白Rv 1980 c）仅在分枝杆菌细胞活跃分裂时表达。凭借这种相对独特的表达谱，Rv 1980 c目前正在南非和秘鲁进行III期临床试验，以评估其替代结核菌素或纯化蛋白衍生物作为活动性结核病感染的基准诊断的潜力。尽管许多NIH赞助的倡议支持结核病研究和Rv 1980 c的潜在重要性，以改善诊断的单一最广泛的细菌感染性疾病在世界上，传统的遗传和生物化学方法未能提供任何解释的生物学作用的Rv 1980 c或任何其他成员的蛋白质家族。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，通过以下具体目标来更好地理解MPT 64家族的生物学作用：具体目标1：通过杂波NMR光谱确定Rv 1980 c的高分辨率溶液结构的集合。具体目标二：通过解析鸟分枝杆菌MPT 64基因组中2个MPT 64家族成员中的1个MAP 0087的高分辨晶体结构，研究MPT 64家族中结构相似和差异的区域。副结核病（地图）。具体目标3：使用异源NMR比较野生型二硫键连接的Rv 1980 c与非连接突变蛋白的结构和动力学性质，以深入了解Rv 1980 c皮肤屏障转运的机制。目的1和2的结果将相互关联，并通过与已知功能的蛋白质的结构同源性将可能的生物学作用分配给MPT 64家族成员;这将进一步加深我们对这些蛋白质在M的复杂发病机制中的理解。结核病和相关生物。目标3的结果将提供Rv 1980 c皮肤屏障转运的机制见解，并可能建议改进这种有前途的诊断剂的方法。这里提出的研究有可能影响广泛的分枝杆菌疾病的治疗和诊断，因此属于该机构的使命。

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.