Summary L eucine R ich R epeat K inase 2 ( LRRK2 ) is the most commonly mutated gene in familial Parkinson’s disease (PD) 1 and is also linked to its idiopathic form 2 . LRRK2 is proposed to function in membrane trafficking 3 and co-localizes with microtubules 4 . Despite LRRK2’s fundamental importance for understanding and treating PD, there is limited structural information on it. Here we report the 3.5Å structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14Å cryo-electron tomography in situ structure 5 . We propose that the conformation of LRRK2’s kinase domain regulates its microtubule interaction, with a closed conformation favoring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin-1 and cytoplasmic dynein-1 in vitro . Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.
Eukaryotic transcription-coupled repair (TCR), or transcription-coupled nucleotide excision repair (TC-NER), is an important and well-conserved sub-pathway of nucleotide excision repair (NER) that preferentially removes DNA lesions from the template strand blocking RNA polymerase II (Pol II) translocation1,2. Cockayne syndrome group B protein in humans (CSB, or ERCC6), or its yeast orthologs (Rad26 in Saccharomyces cerevisiae and Rhp26 in Schizosaccharomyces pombe), is among the first proteins to be recruited to the lesion-arrested Pol II during initiation of eukaryotic TCR1,3–10. Mutations in CSB are associated with Cockayne syndrome, an autosomal-recessive neurologic disorder characterized by progeriod features, growth failure, and photosensitivity1. The molecular mechanism of eukaryotic TCR initiation remains elusive, with several long-standing questions unanswered: How do cells distinguish DNA lesion-arrested Pol II from other forms of arrested Pol II? How does CSB interact with the arrested Pol II complex? What is the role of CSB in TCR initiation? The lack of structures of CSB or the Pol II-CSB complex have hindered our ability to answer those questions. Here we report the first structure of S. cerevisiae Pol II-Rad26 complex solved by cryo-electron microscopy (cryo-EM). The structure reveals that Rad26 binds to the DNA upstream of Pol II where it dramatically alters its path. Our structural and functional data suggest that the conserved Swi2/Snf2-family core ATPase domain promotes forward movement of Pol II and elucidate key roles for Rad26/CSB in both TCR and transcription elongation.
The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein–Lis1 complex. Our 3.1Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.
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