Biochemical And Structural Analysis Of How Parkinson’s Disease-associated Mutations In LRRK2 Protein Cause The Disease

Parkinson’s disease（PD）is one of the most common neurodegenerative disease.Missense mutations in LRRK2（Leucine-rich repeat Kinase2）account for both familial and sporadic Parkinson’s disease.LRRK2 is a muti-functional protein that is comprised of seven domains.The catalytical central architecture of LRRK2 are the Roc-COR tandem domain（Roc protein belongs to the small G proteins）followed by the kinase domain which belongs to the class of serine/threonine protein kinase.Most of the pathogenetic mutations are located in the Roc-COR-kinase tandem domain of LRRK2 protein.The mutations cause an abberant over-activated kinase activity of LRRK2 protein,meanwhile the mutations located in the Roc-COR domain result in an impaired GTPase acitivity as well.Recent studies have shown that the GTPase activity of LRRK2 protein regulates the intramolecular downstream kinase activity.However,the mechanism involved remains unclear.Moreover,it is still obscure whether changes of GTPase activity is linked to kinase activity alteration directly.A recent study has shown that the G-domain of LRRK2 cycles between its monomeric and dimeric conformations upon binding to GTP or GDP,respectively,and that the PD-associated mutations R1441C/G/H impair G-domain monomer-dimer dynamics and are trapped in a constitutive monomeric conformation.That led us to question whether other disease-associated mutations in the G-domain would also affect its conformation.Here,we report that another PD-associated mutation N1437H(Roc_N1437H)also impairs its monomer-dimer conformational dynamic and its GTPase activity.In contrast to mutations at R1441,we found that Roc_N1437H1437H is locked in a stable dimeric conformation in solution and its GTPase activity is about 3.5 fold lower than that of the wild-type.Furthermore,we found that the N1437H mutation causes about 2.5 fold decrease in GTP nucleotide binding affinity compared to other pathogenic mutations in GTPase domain.We further show that GTP dissociation rate（off rate）of Roc_N1437H1437H is slower than wild type,indicating that N1437H might interrupt the process of nucleotide exchange cycle.Taken together,our data support that conformational dynamics is important for the GTPase activity of LRRK2 and that the disease-associated mutation N1437H impairs GTPase activity by locking the ROC domain in a persistently dimeric state.In Addition,recent studies have reported that the R1398H variant in Roc domain is suggested in genetic studies to confer protection against PD but the mechanism is still lacking.Here,we investigated the biochemical characteristics of R1398H mutant to understand the underlying mechanism.We found that there was no significant difference in the conformation,thermal stability,secondary structure and GTP binding ability of Roc_R1398H1398H compared with Roc_WT.But the GTPase activity of Roc_R1398H1398H was lower than one of the wild type,and the GDP binding affinity of Roc_R1398H1398H was higher than Roc_WT.We have solved the crystal structure of GDP-bound R1398H.However,based on the analysis of structural comparsion,there was no obviouse difference between R1398H and the wild type.