Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2

Reviewer #1 (Public review):

Summary:

In this manuscript, Chen et al. used cryo-ET and in vitro reconstituted system to demonstrate that the autoinhibited form of LRRK2 can also assemble into filaments that wrap around the microtubule, although the filaments are typically shorter and less regular compared to the previously reported active-LRRK2 filaments. The structure revealed a new interface involving the N-terminal repeats that were disordered in the previous active-LRRK2 filament structure. The autoinhibited-LRRK2 filament also has different helical parameters compared to the active form.

Strengths:

The structure obtained in this study is the highest resolution of LRRK2 filaments done by subtomogram averaging, representing a major technical advance compared to the previous Cell paper from the same group. Overall, I think the data are well presented with beautiful graphic rendering, and valuable insights can be gained from this structural study.

Weaknesses:

(1) There are only three main figures, together with 9 supplemental figures. The authors may consider breaking the currently overwhelming Figures 1 and 3 into smaller figures and moving some of the supplemental figures to the main figure, e.g., Figure S7.

(2) The key analysis of this manuscript is to compare the current structure with the previous active-LRRK2 filament structure. Currently, such a comparison is buried in Figure 3H. It should be part of Figure 1.

Reviewer #2 (Public review):

The authors of this paper have done much pioneering work to decipher and understand LRRK2 structure and function, to uncover the mechanism by which LRRK2 binds to microtubules, and to study the roles that this may play in biology. Their previous data demonstrated that LRRK2 in the active conformation (pathogenic mutation or Type I inhibitor complex) bound to microtubule filaments in an ordered helical arrangement. This they showed induced a "roadblock" in the microtubule impacting vesicular trafficking. The authors have postulated that this is a potentially serious flaw with Type 1 inhibitors and that companies should consider generating Type 2 inhibitors in which the LRRK2 is trapped in the inactive conformation. Indeed the authors have published much data that LRRK2 complexed to Type 2 inhibitors does not seem to associate with microtubules and cause roadblocks in parallel experiments to those undertaken with type 1 inhibitors published above.

In the current study, the authors have undertaken an in vitro reconstitution of microtubule-bound filaments of LRRK2 in the inactive conformation, which surprisingly revealed that inactive LRRK2 can also interact with microtubules in its auto-inhibited state. The authors' data shows that while the same interphases are seen with both the active LRRK2 and inactive microtubule bound forms of LRRK2, they identified a new interphase that involves the WD40-ARM-ANK- domains that reportedly contributes to the ability of the inactive form of LRRK2 to bind to microtubule filaments. The structures of the inactive LRRK2 complexed to microtubules are of medium resolution and do not allow visualisation of side chains.

This study is extremely well-written and the figures are incredibly clear and well-presented. The finding that LRRK2 in the inactive autoinhibited form can be associated with microtubules is an important observation that merits further investigation. This new observation makes an important contribution to the literature and builds upon the pioneering research that this team of researchers has contributed to the LRRK2 fields. However, in my opinion, there is still significant work that could be considered to further investigate this question and understand the physiological significance of this observation.

Reviewer #3 (Public review):

Summary:

The manuscript by Chen et al examines the structure of the inactive LRRK2 bound to microtubules using cryo-EM tomography. Mutations in this protein have been shown to be linked to Parkinson's Disease. It is already shown that the active-like conformation of LRRK2 binds to the MT lattice, but this investigation shows that full-length LRRk2 can oligomerize on MTs in its autoinhibited state with different helical parameters than were observed with the active-like state. The structural studies suggest that the autoinhibited state is less stable on MTs.

Strengths:

The protein of interest is very important biomedically and a novel conformational binding to microtubules in the proposed.

Weaknesses:

(1) The structures are all low resolution.

(2) There are no measurements of the affinity of the various LRRK2 molecules (with and without inhibitors) to microtubules. This should be addressed through biochemical sedimentation assay.