Indiana University-Purdue University Indianapolis (IUPUI) / Mutations in the gene encoding for leucine rich repeats kinase 2 (LRRK2) are commonly found in Parkinson’s disease. Recently, we found that the disease-associated point mutations at residue R1441 in the G domain (ROC) of LRRK2 resulted in perturbation of its GTPase activity. In this study, we compare the biochemical and biophysical properties of the ROC domain of LRRK2 carrying the PD-associated mutations at residue R1441 with those of the wild-type. We found that the disease-associated mutations (R1441C/G/H) showed marked quaternary structure compared to wild-type, in that the latter existed in solution in both monomeric and dimeric conformations dynamically regulated by GDP/GTP binding state, while we detected only monomeric conformation for three disease-associated mutants. To understand the structural basis for this plasticity and the activity reduction in the mutants, we solved a 1.6 Å crystal structure of the wild type ROC that shows a stable dimeric conformation in which the switch motifs and inter-switch regions mediate extensive interactions at the dimer interface. Residue R1441, where PD-associated mutations occur, forms exquisite interactions at the interface, thus suggesting a critical role of this residue in maintaining a dynamic dimer-monomer interconversion and conformational flexibility of the switch motifs. Consistently, substituting R1441 for other arbitrary mutations (R1441K/S/T) lead to similar perturbation of GTPase activity and dimerization defects as observed in the disease-associated mutants. Locking the ROC domain in either dimeric or monomeric conformations by engineered disulfide bond alters the binding affinity to GTP (but not GDP) and significantly reduce GTPase activity, thus suggesting that the dynamic dimer-monomer interconversion and conformational plasticity are essential for ROC function as a molecular switch modulating the kinase activity of LRRK2.
| Identifer | oai:union.ndltd.org:IUPUI/oai:scholarworks.iupui.edu:1805/20692 |
| Date | 08 1900 |
| Creators | Wu, Chunxiang |
| Contributors | Hoang, Quyen Q., Foroud, Tatiana M., Hurley, Thomas D., Johnson, Steven M., Zhang, Zhong-Yin |
| Source Sets | Indiana University-Purdue University Indianapolis |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
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