Role of protein A structure, folding kinetics and dynamics in S. aureus virulence

 描述(申请人提供)：葡萄球菌蛋白A(SPA)是人类重要的细菌病原体金黄色葡萄球菌的主要毒力因子。它拥有大量不同的功能，主要是通过它与 宿主免疫和炎症反应系统。该提案的目的在于确定这些活动的结构性和机械性起源，最终目标是治疗干预。对传统抗生素和“最后手段”抗生素产生抗药性的社区获得性金黄色葡萄球菌感染的出现，对人类健康构成了重大且日益严重的威胁。许多比较野生型菌株和缺乏SpA菌株的研究已经证实，这种细胞表面蛋白在细菌逃避免疫系统的能力中发挥着重要作用，并导致炎性脓毒症，后者是患者死亡的最终原因。我们建议通过X射线结晶学测定抗体片段(Fc)复合体中SpA和/或其结构域的结构来研究这两种活性。先前的研究已经证实，该蛋白质的功能部分是高度灵活的，由五个几乎相同的结构域组成，每个结构域都具有形成上述复合体的能力。我们开发了一种复杂的方法来描述磁区间取向分布(IOD)，该方法基于两个磁区结构中剩余的偶极耦合测量，其中一个磁区通过稀土元素结合标签与磁场对准。我们计划比较存在和不存在单体Fc时的IOD，以更好地了解IOD和抗体结合的相互依赖。我们还计划通过移除相邻结构域的螺旋3的C-末端帽和螺旋1的N-末端帽来构建SPA和各种多结构域的“刚性”版本。如果我们成功地生产出这样一种刚性蛋白质，我们将研究强化化对功能的影响，包括抗体结合和细胞表面受体激活。为了发挥其各种功能，蛋白质必须通过细胞膜转运，并附着在细胞壁的肽聚糖骨架上。我们建议研究SpA的分泌机制，特别是蛋白质N-末端半部分的快速去折叠/再折叠(RUF)特性的作用。对SpA分泌的更好了解将有助于开发阻断分泌的治疗方法，从而消除其所有有毒功能。综上所述，拟议的研究为金黄色葡萄球菌使用的一种关键武器的结构和功能提供了广泛的生物物理见解，使其成为美国对人类健康构成的两到三个最重要的细菌威胁之一。为了获得这些洞察力，我们计划开发几种新的生物物理方法，这些方法将应用于其他柔性和RUF蛋白质。

 DESCRIPTION (provided by applicant): Staphylococcal protein A (SpA) is a major virulence factor of the important human bacterial pathogen, Staphylococcus aureus. It possesses a surprisingly large range of disparate functions, primarily through its interaction with elements of host immune and inflammatory response systems. The aims of this proposal seek to define the structural and mechanistic origins of these activities with the ultimate goal of therapeutic intervention. The emergence of community acquired S. aureus infections resistant to traditional and "last-resort" antibiotics is a significant and rising threat to human health. Many studies comparing wild-type strains to those lacking SpA have established that this cell surface protein plays an important role in the bacterium's ability to evade the immune system and contribute to the inflammatory sepsis that is the ultimate cause of patient death. We propose to study both of these activities by determining the structures of SpA and/or its domains in complex with antibody fragments (Fc) by X-ray crystallography. Previous studies have established that the functional half of the protein is highly flexible and consists of five nearly identical domains tha each possesses the ability to form the complexes described above. We have developed a sophisticated method to describe the interdomain orientational distributions (IOD) based on residual dipolar coupling measurements in two-domain constructs in which one domain is aligned with the magnetic field via a lanthanide binding tag. We plan to compare the IOD in the presence and absence of monomeric Fc to better understand the interdependence of the IOD and antibody binding. We also plan to construct a "rigid" version of SpA and various multidomain constructs by removing the C- terminal cap of helix 3 and the N-terminal cap of helix 1 of the adjacent domain. If we are successful in producing such a rigid protein, we will study the effect of rigidification on function, including antibody binding and cell surface recepto activation. To serve its various functions, the protein must be translocated through the cell membrane and attached to the peptidoglycan framework of the cell wall. We propose to study the mechanism of SpA secretion, particularly the role of the rapid unfolding/refolding (RUF) property of the N-terminal half of the protein. A better understanding of SpA secretion would allow the development of therapeutic approaches to blocking secretion, thereby eliminating all of its virulent functions. Taken together, the proposed studies offer a broad range of biophysical insights into the structure and function of a key weapon used by S. aureus to make it one of the two or three most significant bacterial threats to human health in the US. To obtain these insights, we plan to develop several new biophysical methods that will have applications to other flexible and RUF proteins.