Structural insights into the mechanism of the novel dimeric ROC GTPase from LRRK2

描述（由申请人提供）：富亮氨酸重复激酶2 （LRRK2）突变是帕金森病（PD）的最常见原因。LRRK2含有一个Ras of complex proteins （ROC）结构域，可以作为GTP酶，通过GTP/GDP结合循环调节其蛋白激酶活性。GTP被ROC结构域水解的机制以及它如何调节激酶活性尚不清楚。目前迫切需要更好地了解LRRK2的结构/功能，以阐明其在PD发展中的作用。本提案的重点是通过独特的二聚体ROC结构域提供对GTP水解机制的结构见解，以及内在的ROC/COR结构域关联。我们的主要假设是LRRK2作为二聚体发挥作用，而独特的二聚体结构域交换的ROC GTPase结构域通过COR结构域作为分子铰链调节激酶活性。我们将使用x射线晶体学结合其他生物物理和生化方法来：1)进一步表征二聚体ROC结构域，特别是二聚体在GTP结合袋外区域的稳定性对GTP酶活性的影响。2)阐明活动状态下ROC域的结构。3).在ROC域上映射COR结合区域。我们的长期目标是了解LRRK2的功能。本应用程序的目标是，这是追求这一目标的下一步，是在ROC结构域中详细表征新型二聚体GTP酶，以更好地了解其GTP水解机制和在调节激酶活性中的作用。预期结果将揭示GTP/GDP结合周期中蛋白质的运动和修饰，以及相关PD相关突变的分子基础。这项研究获得了额外的维度。由于靶向蛋白具有重要的医学相关性，我们的研究将为设计选择性抑制剂/激活剂提供一个平台，这些抑制剂/激活剂可能反过来进一步开发成针对PD的新疗法。我们提议的研究与NIH的使命密切相关，即帮助促进人类健康和延长寿命。我们的研究对加强俄克拉荷马州立大学的生物医学研究具有更广泛的影响，该大学迄今为止还不是NIH资助的主要接受者。拟议的研究和使用的先进技术将吸引越来越多的本科生和研究生从事生物医学研究，并为该地区创造更有活力和竞争的研究环境。作为俄克拉何马州为数不多的新晶体学家之一，PI站在一个独特的位置，吸引各种背景的学生。这笔资金将用于招收兰斯顿大学的少数民族学生，并对他们进行x射线晶体学方面的培训。兰斯顿大学是一所历史悠久的非裔美国人本科院校，距离俄克拉荷马州立大学约20英里。希望这里提出的新的x射线晶体学技术和具有很强生物医学相关性的非常有趣的项目能够吸引更多的本科生从事生物医学研究。公共卫生相关性：拟议的研究具有重要意义，与公共卫生相关，因为预计将在疾病治疗和延长人类寿命方面取得重大进展。此外，所了解的内容有望有助于更好地理解细胞信号通路的分子机制。拟议的研究将刺激俄克拉何马州更强大和更具竞争力的生物医学研究环境。

DESCRIPTION (provided by applicant): Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may act as a GTPase to regulate its protein kinase activity through a GTP/GDP bound cycle. The mechanisms of the GTP hydrolysis by the ROC domain and how it regulates the kinase activity are not known. There is a critical need to better understand the structure/function of LRRK2 to elucidate its roles in PD development. The focus of this proposal is on providing structural insights into the mechanism of the GTP hydrolysis by the unique dimeric ROC domain, as well as the intrinsic ROC/COR domain associations. Our central hypotheses are that LRRK2 function as a dimer and the unique dimeric domain-swapped ROC GTPase domain regulates the kinase activity, via the COR domain as a molecular hinge. We will use x-ray crystallography in combination with other biophysical and biochemical methods to: 1). further characterize the dimeric ROC domain, in particular, the effect of dimer stabilities in the regions outside of the GTP binding pocket, on the GTPase activity. 2). elucidate the structure of the ROC domain in the active state. 3). map the COR binding regions on the ROC domain. Our long-term goal is to understand how LRRK2 functions. The objective of this application, which is the next step in pursuit of that goal, is the detailed characterization of the novel dimeric GTPase in the ROC domain to better understand its mechanism of GTP hydrolysis and roles in regulating the kinase activity. The expected results will reveal the motions and modifications of the protein during the GTP/GDP bound cycle, as well as the molecular basis for the related PD associated mutations. This research obtains/assumes extra dimensions. Since the targeted proteins are of significant medical relevance, our studies will provide a platform for designing selective inhibitors/activators that may in their turn be further developed into new therapeutics against PD. Our proposed research is closely relevant to NIH's mission to help promote better human health and longer life-span. Our research has broader impacts on strengthening the biomedical research in Oklahoma State University, which has not been a major recipient of NIH funding to date. The proposed research and the advanced techniques to be used will attract more and more undergraduate and graduate students to biomedical research and stimulate more vigorous and competitive research environment in the region. As a new and one of few crystallographers here in Oklahoma, the PI stands in a unique position to attract students with a wide variety of backgrounds. Funds from this grant will be used to recruit minority students from Langston University and train them in X-ray crystallography. Langston University is a historically African- American undergraduate institution located ~20 miles away from Oklahoma State University. It is expected that the new x-ray crystallographic techniques and the very interesting projects with strong biomedical relevance proposed here will attract more undergraduate students to biomedical research. PUBLIC HEALTH RELEVANCE: The proposed research is significant and relevant to public health, because important advances in the therapy of diseases and prolongation of human life span would be expected. In addition, what is learned is expected to contribute to better understanding the molecular mechanism in cell signaling pathway. The proposed studies will stimulate stronger and more competitive biomedical research environment in Oklahoma.