Intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism

Reviewer #1 (Public Review):

Summary:

This study presents careful biochemical experiments to understand the relationship between LRRK2 GTP hydrolysis parameters and LRRK2 kinase activity. The authors report that incubation of LRRK2 with ATP increases the KM for GTP and decreases the kcat. From this they suppose an autophosphorylation process is responsible for enzyme inhibition. LRRK2 T1343A showed no change, consistent with it needing to be phosphorylated to explain the changes in G-domain properties. The authors propose that phosphorylation of T1343 inhibits kinase activity and influences monomer-dimer transitions.

Strengths:

Strengths of the work are the very careful biochemical analyses and interesting result for wild type LRRK2.

Weaknesses:

The conclusions related to involvement of a monomer-dimer transition are to this reviewer, premature and an independent method needs to be utilized to bolster this aspect of the story.

Reviewer #2 (Public Review):

As discussed in the original review, this manuscript is an important contribution to a mechanistic understanding of LRRK2 kinase. Kinetic parameters for the GTPase activity of the ROC domain have been determined in the absence/presence of kinase activity. A feedback mechanism from the kinase domain to GTP/GDP hydrolysis by the ROC domain is convincingly demonstrated through these kinetic analyses. However, a regulatory mechanism directly linking the T1343 phospho-site and a monomer/dimer equilibrium is not fully supported. The T1343A mutant has reduced catalytic activity and can form similar levels of dimer as WT. The revised manuscript does point out that other regulatory mechanisms can also play a role in kinase activity and GTP/GDP hydrolysis (Discussion section). The environmental context in cells cannot be captured from the kinetic assays performed in this manuscript, and the introduction contains some citations regarding these regulatory factors. This is not a criticism, the detailed kinetics here are rigorous, but it is simply a limitation of the approach. Caveats concerning effects of membrane localization, Rab/14-3-3 proteins, WD40 domain oligomers, etc... should be given more prominence than a brief (and vague) allusion to 'allosteric targeting' near the end of the Discussion.

Specific comments

(1) The revised version is better organized with respect to the significance of monomer/dimer equilibrium and the relevance of the GTP-binding region of ROC domain that encompasses the T1343 phospho-site. The relevance of monomers/dimers of LRRK2 from previous studies is better articulated and readers are able to follow the reasoning for the various mutations.

(2) As a suggestion I would change the following on page 6 to clarify for readers:
"...would show no change in kcat and KM values upon in vitro ATP treatment" to:
"...would show no change in kcat and KM values for GTP hydrolysis upon in vitro ATP treatment"

(3) The levels of dimer in WT (+ATP) and T1343A (+/- ATP) are the same, about 40-45%. These data are cited when the authors state that ATP-induced monomerization is 'abolished' (page 6). My suggestion is to re-phrase this conclusion for consistency with data (Fig 5). For example, one can state that 'ATP incubation does not affect the percentage of dimer for the T1343A variant of LRRK2'. This would be similar to the authors' description of these data on page 8 - 'no difference in dimer formation upon ATP treatment'.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

This study presents careful biochemical experiments to understand the relationship between LRRK2 GTP hydrolysis parameters and LRRK2 kinase activity. The authors report that incubation of LRRK2 with ATP increases the KM for GTP and decreases the kcat. From this, they suppose an autophosphorylation process is responsible for enzyme inhibition. LRRK2 T1343A showed no change, consistent with it needing to be phosphorylated to explain the changes in G-domain properties. The authors propose that phosphorylation of T1343 inhibits kinase activity and influences monomer-dimer transitions.

Strengths:

The strengths of the work are the very careful biochemical analyses and the interesting result for wild-type LRRK2.

Weaknesses:

A major unexplained weakness is why the mutant T1343A starts out with so much lower activity--it should be the same as wild-type, non-phosphorylated protein. Also, if a monomer-dimer transition is involved, it should be either all or nothing. Other approaches would add confidence to the findings.

We thank the reviewer for these suggestions. We are aware that the T1343A has generally a lower activity compared to the wild type. Therefore, we would like to emphasize that this mutant is the only one not showing an increase in Km values after ATP treatment. Other mutants, also having lower kcat values like T1503A, still show this characteristic change in Km. Our favored explanation for the lower kcat of T1343A is that this mutation lays within a critical region, the so-called ploop, of the Roc domain and is very likely structurally not neutral. Concerning the dimer-monomer transition, we are convinced that there is more than one factor involved in this equilibrium. Most likely, including, but not limited to other LRRK2 domains (e.g. the WD40 domain), binding of co-factors (e.g. Rab29/Rab32 or 14-3-3) and membrane binding. Consistently, also with stapled peptides targeting the Roc or Cor domains we were not able to shift the equilibrium completely to the monomer (Helton et al., ACS Chem Biol. 2021, 16:2326-2338; Pathak et al. ACS Chem Neurosci. 2023, 14(11):1971-1980) We have addressed these points in a revised version of the manuscript.

Reviewer #2 (Public Review):

This study addresses the catalytic activity of a Ras-like ROC GTPase domain of LRRK2 kinase, a Ser/Thr kinase linked to Parkinson's disease (PD). The enzyme is associated with gain-of-function variants that hyper-phosphorylate substrate Rab GTPases. However, the link between the regulatory ROC domain and activation of the kinase domain is not well understood. It is within this context that the authors detail the kinetics of the ROC GTPase domain of pathogenic variants of LRRK2, in comparison to the WT enzyme. Their data suggest that LRRK2 kinase activity negatively regulates the ROC GTPase activity and that PD variants of LRRK2 have differential effects on the Km and catalytic efficiency of GTP hydrolysis. Based on mutagenesis, kinetics, and biophysical experiments, the authors suggest a model in which autophosphorylation shifts the equilibrium toward monomeric LRRK2 (locked GTP state of ROC). The authors further conclude that T1343 is a crucial regulatory site, located in the P-loop of the ROC domain, which is necessary for the negative feedback mechanism. Unfortunately, the data do not support this hypothesis, and further experiments are required to confirm this model for the regulation of LRRK2 activity.

Specific comments are below:

• Although a couple of papers are cited, the rationale for focusing on the T1343 site is not evident to readers. It should be clarified that this locus, and perhaps other similar loci in the wider ROCO family, are likely important for direct interactions with the GTP molecule.

To clarify this point: We, have not only have focused on this specific locus, but instead systematically mutated all known auto-phosphorylation sites with the RocCOR domain (see. supplemental information). Furthermore, it has been shown that this site, at least in the RCKW (Roc to WD40) construct, is quantitatively phosphorylated (Deniston et al., Nature 2020, 588:344-349). We are aware that the T1343 residue is located within the p-loop and that this can impact nucleotide binding capacities (see response to reviewer 1).

We have clarified and addressed these points in a revised version of the manuscript.

• Similar to the above, readers are kept in the dark about auto-phosphorylation and its effects on the monomer/dimer equilibrium. This is a critical aspect of this manuscript and a major conceptual finding that the authors are making from their data. However, the idea that auto-phosphorylation is (likely) to shift the monomer/dimer equilibrium toward monomer, thereby inactivating the enzyme, is not presented until page 6, AFTER describing much of their kinetics data. This is very confusing to readers, as it is difficult to understand the meaning of the data without a conceptual framework. If the model for the LRRK2 function is that dimerization is necessary for the phosphorylation of substrates, then this idea should be presented early in the introduction, and perhaps also in the abstract. If there are caveats, then they should be discussed before data are presented. A clear literature trail and the current accepted (or consensus) mechanism for LRRK2 activity is necessary to better understand the context for these data.

We agree on the reviewer’s opinion. We have revised the introduction accordingly and added a paragraph on page 3 starting from line 27.

• Following on the above concepts, I find it interesting that the authors mention monomeric cytosolic states, and kinase-active oligomers (dimers??), with citations. Again here, it would be useful to be more precise. Are dimers (oligomers?) only formed at the membrane? That would suggest mechanisms involving lipid or membrane-attached protein interactions. Also, what do the authors mean by oligomers? Are there more than dimers found localized to the membrane?

There are multiple studies that have shown that LRRK2 is mainly monomeric in the cytosol while it forms mainly dimeric or higher oligomeric states at membrane (James et al., Biophys. J. 2012, 102, L41–L43; Berger et al., Biochemistry, 2010, 49, 5511–5523). However, we agree with the reviewer that it remains to be determined if the dimeric form is the most active state at the membrane, or a higher oligomeric state. Espescially since a recent study shows that LRRK2 can form active tetramers only when bound to Rab29 (Zhu et al., bioRxiv, 2022, DOI: 10.1101/2022.04.26.489605). We have clarified these points in the introduction of the revised version of the manuscript (page 3, line 27ff).

• Fig 5 is a key part of their findings, regarding the auto-phosphorylation induced monomer formation of LRRK2. From these two bar graphs, the authors state unequivocally that the 'monomer/dimer equilibrium is abolished', and therefore, that the underlying mechanism might be increased monomerization (through maintenance of a GTP-locked state). My view is that the authors should temper these conclusions with caveats. One is that there are still plenty of dimers in the auto-phosphorylated WT, and also in the T1343A mutant. Why is that the case? Can the authors explain why only perhaps a 10% shift is sufficient? Secondly, the T1343A mutant appears to have fewer overall dimers to begin with, so it appears to readers that 'abolition' is mainly due to different levels prior to ATP treatment at 30 deg. I feel these various issues need to be clarified in a revised manuscript, with additional supporting data. Finally, on a minor note, I presume that there are no statistically significant differences between the two sets of bar graphs on the right panel. It would be wise to place 'n.s.' above the graphs for readers, and in the figure legend, so readers are not confused.

Starting with the monomer-dimer equilibrium we are convinced that there is more than the phosphorylation of T1343 (see response to reviewer 1). Therefore a 10% shift in our assay most likely underestimate the effect seen in cells. Consistently, the T1343A mutants show a similar increase in Rab10 phosphorylation assay as the G2019S mutant. This thus shows that the identified feedback mechanism plays an important role in a cellular context. We have addressed this point in the revised manuscript on page 6, line 8ff. As long as the significance indicators in the bar charts are concerned, we agree with reviewer. In order not to overload the figure, we finally decided to include all pairwise comparisons (post-hoc tests) in the supplement.

• Figure 6B, Westerns of phosphorylation, the lanes are not identified and it is unclear what these data mean.

We apologize for this mistake and have added the correct labeling in the revised version of the manuscript.