Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.LRRK2 | Parkinson's disease | structural modeling | EM | CL-MS

Mutations in LRRK2 are a common cause of genetic Parkinson’s disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2.

Purine nucleosides on position 9 of eukaryal and archaeal tRNAs are frequently modified in vivo by the post-transcriptional addition of a methyl group on their N1 atom. The methyltransferase Trm10 is responsible for this modification in both these domains of life. While certain Trm10 orthologues specifically methylate either guanosine or adenosine at position 9 of tRNA, others have a dual specificity. Until now structural information about this enzyme family was only available for the catalytic SPOUT domain of Trm10 proteins that show specificity toward guanosine. Here, we present the first crystal structure of a full length Trm10 orthologue specific for adenosine, revealing next to the catalytic SPOUT domain also N- and C-terminal domains. This structure hence provides crucial insights in the tRNA binding mechanism of this unique monomeric family of SPOUT methyltransferases. Moreover, structural comparison of this adenosine-specific Trm10 orthologue with guanosine-specific Trm10 orthologues suggests that the N1 methylation of adenosine relies on additional catalytic residues.