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.
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.
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