LRRK2 Mediated Phosphorylation Increases NSF ATPase Activity

Once identified that NSF is a substrate of LRRK2 kinase activity, the next step was to investigate whether this phosphorylation has functional consequences on the ATPase activity of NSF. In contrast to NSF, purification of recombinant LRRK2 from HEK293T cells requires the presence of Tween-20 in the lysis buffer. One possibility is that LRRK2 is stabilized by the detergent, as it has been shown to interact with membranous structures inside the cell. We first identified the optimal reaction conditions at which both NSF and LRRK2 display their maximal activity, since we needed to pre-incubate NSF with LRRK2 (or only buffer) to allow phosphorylation, before proceeding with the ATPase assays. The activity of NSF was evaluated with the Malachite Green enzyme

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is preserved at a low concentration of detergent, therefore the subsequent assays were performed with the presence of Tween-20 at its CMC concentration.

Fig.7.8.1 | Evaluation of the optimal detergent concentration: Optimal Tween-20 concentration

for the reactions was evaluate within ability of LRRK2 to phosphorylate itself measuring the intensity of the band with an antibody against one of its auto-phosphorylation site (Thr-2483). At the same time, the activity of human NSF, using the same concentration of detergent, was evaluate measuring the V0 of the reaction with the Malachite Green Enzyme assay.

NSF was treated with LRRK2-G2019S970-2527 _{at a ratio NSF:LRRK2 = 20:1 or alone in the}

kinase buffer at 30°C for 1 hour and 30 minutes in the presence of 50 μM ATP which is sufficient to support LRRK2 phosphorylation and minimizes interference with the subsequent ATPase assay. We incubated 500 nM of NSF (monomer) and then performed ATPase assays at a final NSF concentration of 36 nM (hexameric) based on previous studies that employed this concentration to monitor NSF activity (Cipriano et al., 2013; Vivona et al., 2013). We used the Malachite Green enzyme assay, to measure the kinetic parameters of NSF and to evaluate if they change upon LRRK2 phosphorylation. Pi

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release over time was measured in NSF-phosphorylated and NSF-unphosphorylated. As summarized in Fig.7.8.2, we found that the kinetic parameters of the reaction calculated for recombinant human flag-tagged NSF increased of about 2-fold upon LRRK2 phosphorylation.

Fig.7.8.2 | LRRK2 phosphorylation increase the ATPase activity of NSF: (A)NSF ATPase activity

was assessed with a Malachite green assays at 36 nM NSF (hexamer) at different ATP concentration to calculate the kinetic parameters of NSF (red, NSF) and NSF phosphorylated by LRRK2-G2019S970-2527 _{(blue, NSF-LRRK2 ΔG2019S). Malachite Green assay was repetead for}

LRRK2-G2019S970-2527 _{(pink, LRRK2 ΔG2019S) as a control. Data were fitted with the Michaelis-}

Menten kinetic model to determine kinetic constants (n=3). (B) Kinetic parameters obtained from the experiment show in A. Data reveal that NSF increases its activity within 2-fold upon LRRK2 ΔG2019S phosphorylation.

We then repeated the experiment in presence of the different NSF mutants and 1.4 mM ATP to evaluate whether the abolishment of the phosphorylation site would prevent the observed increased ATP activity observed upon LRRK2 phosphorylation. Interestingly we found that both T645A and T646A mutants display a low basal activity independently of phosphorylation, whereas S647A mutant has a similar ATPase activity compared to wild- type form (Fig.7.8.3). These results suggest that T645 and T646 residues may play a key role in NSF activity, since mutations into alanine provoke impairment of ATPase activity.

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Fig.7.8.3 | ATPase activity of NSF wild-type and all mutants upon LRRK2 phosphorylation: Pi

generated by ATP hydrolysis in the presence of NSF wild-type, NSFT645A _NSFT646A _NSFS647A _pre-

phosphorylated or not by LRRK2 ΔG2019S (P-) was measured with the Malachite Green Assay at 120 min with an initial concentration of ATP of 1.4 mM (NSFWT _{vs NSF}T645A _{**p<0.01; NSF}WT _{vs P-}

NSFWT _{***p<0.001; NSF}T645A _{vs P-NSF}T645A _{p>0.05, not significant, n.s.; NSF}T646A _{vs P-NSF}T646A

p>0.05, n.s.; NSFS647A _{vs P-NSF}S647A _{**p<0.01; one-way ANOVA, Bonferroni’s post-test, n≥3).}

UT=untrasfected cells subjected to Flag-affinity purification to monitor background activity (n=1; excluded from the statistical analysis).

To rule out the possibility that these differences in the activity of NSF mutants may be due to an incorrect folding of the protein, we compared the circular dichroism (CD) and fluorescence properties of NSF wild-type, T645A, T646A and S647A mutants. As shown in Fig.7.8.4 B, all the proteins exhibit similar CD spectra showing a prevalent alpha-helix conformation. Moreover, intrinsic fluorescence values are also similar without differences in wavelength of the maximum emission value of the tryptophan peak (Fig.7.8.4 A).

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Fig.7.8.4 | Intrisic fluorescence and CD spectra of NSF wild-type and mutants: (A) Protein

concentration used for Intrinsic Fluorescence measurements was 0.9 µM (Ec = 295 nm; Em = 330 nm). (B) CD spectra were normalized by protein concentration previously measured with an SDS- PAGE gel and reveal that all proteins are predominantly folded in alpha-helix conformation.

One final piece of evidence is that both T645A and T646A mutants are both able to form hexamers in solution as indicated by Transmission Electron Microscopy (TEM) in Fig.7.8.6. These results show that the alanine mutation in Thr-645 (as well as in the adjacent residue) does not alter the overall folding and the ability of NSF to goes from monomeric conformation.

Fig.7.8.6 | NSF mutant form hexamers in solution: TEM micrograph of both NSF T645A and

T646A mutant with 1 mM ATP and 4 mM MgCl2. Images reveal the typical hexameric structures

showed above for NSF wild-type in Fig.7.2.2 (scalebar 50 nm).

We then looked at the position of these sites within the C. griseus NSF structure, which presents a 98% amino acid identity with the human sequence and all the three residues are conserved among the two species (Fig.7.2.1). The peptide 639_KLLIIGTTSR648 _{is outside}

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Fig.7.8.5 | 3D structure of NSF D2 domain complexed with ATP: ATP-dependent

oligomerization D2 domain of NSF upon ATP (green) binding. D2 quaternary structure was solved using X-ray diffraction method with a resolution of 1.9 Å (PDB code: 1NSF). In figure residues T645-T646-S647 are yellow-labelled within the monomer (dark red).

Taken together, these data indicate that LRRK2 phosphorylation increase NSF ATPase activity and that T645 is a key site for the enzymatic activity of NSF.

7.9 Phosphorylation Increases Also The SNARE Complex