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

 描述（由申请方提供）：葡萄球菌蛋白A（SpA）是重要的人类细菌病原体金黄色葡萄球菌的主要毒力因子。它拥有令人惊讶的大量不同的功能，主要是通过与 宿主免疫和炎症反应系统。该提案的目的是寻求确定这些活动的结构和机制起源，最终目标是治疗干预。社区的出现使S.对传统和“最后手段”的抗生素具有抗药性的金黄色葡萄球菌感染是对人类健康的重大和日益严重的威胁。许多比较野生型菌株与缺乏SpA的菌株的研究已经确定，这种细胞表面蛋白在细菌逃避免疫系统的能力中起着重要作用，并导致炎症性脓毒症，这是患者死亡的最终原因。我们建议通过X射线晶体学确定SpA和/或其结构域与抗体片段（Fc）复合的结构来研究这两种活性。以前的研究已经确定，蛋白质的功能性一半是高度灵活的，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物的能力。我们已经开发出一种复杂的方法来描述域间取向分布（IOD）的基础上残留的偶极耦合测量在两个域的结构，其中一个域是通过一个镧系元素结合标签与磁场对齐。我们计划在存在和不存在单体Fc的情况下比较IOD，以更好地理解IOD和抗体结合的相互依赖性。我们还计划通过去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的“刚性”版本和各种多结构域构建体。如果我们成功地生产出这种刚性蛋白质，我们将研究刚性化对功能的影响，包括抗体结合和细胞表面受体活化。为了发挥其各种功能，蛋白质必须穿过细胞膜并附着在细胞壁的肽聚糖框架上。我们建议研究SpA分泌的机制，特别是蛋白质N-末端一半的快速展开/重折叠（folding）特性的作用。更好地了解SpA分泌将允许开发阻断分泌的治疗方法，从而消除其所有毒性功能。总的来说，拟议的研究提供了一个广泛的生物物理见解的结构和功能的一个关键武器使用的S。金黄色葡萄球菌，使其成为美国对人类健康威胁最大的两三种细菌之一。为了获得这些见解，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蛋白和非柔性蛋白。

 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.