Kinetic, mechanistic, and structural modeling studies of truncated wild-type leucine-rich repeat kinase 2 and the G2019S mutant

Abstract

Leucine-rich repeat kinase 2 (LRRK2), a large and complex protein that possesses two enzymatic properties, kinase and GTPase, is one of the major genetic factors in Parkinson's disease (PD). Here, we characterize the kinetic and catalytic mechanisms of truncated wild-type (t-wt) LRRK2 and its most common mutant, G2019S (t-G2019S), with a structural interpretation of the kinase domain. First, the substitution of threonine with serine in the LRRKtide peptide results in a much less efficient substrate as demonstrated by a 26-fold decrease in k(cat) and a 6-fold decrease in binding affinity. The significant decrease in k(cat) is attributed to a slow chemical transfer step as evidenced by the inverse solvent kinetic isotope effect in the proton inventory and pL (pH or pD)-dependent studies. The shape of the proton inventory and pL profile clearly signals the involvement of a general base (pK(a) = 7.5) in the catalysis with a low fractionation factor in the ground state. We report for the first time that the increased kinase activity of the G2019S mutant is substrate-dependent. Homology modeling of the kinase domain (open and closed forms) and structural analysis of the docked peptide substrates suggest that electrostatic interactions play an important role in substrate recognition, which is affected by G2019S and may directly influence the kinetic properties of the enzyme. Finally, the GTPase activity of the t-G2019S mutant was characterized, and the mutation modestly decreases GTPase activity without significantly affecting GTP binding affinity.

Figure 1

Steady-state kinetic studies of LRRK2-catalyzed phosphorylation. (A) Phosphorylation of LRRKtide by t-wt LRRK2 (○) and the mutant t-G2019S (●). (B) Phosphorylation of LRRKtide^S by t-wt LRRK2 (●) and the mutant t-G2019S (○). (C) Phosphorylation of PLK-peptide by t-wt LRRK2 (●) and the mutant t-G2019S (○). (D) Structural details of peptide binding (LRRKtide on the top-left and PLK-peptide on the top-right) near the ATP binding site of LRRK2. Both peptides show a series of conserved interactions involving residues such as D1887 and R1915. In case of LRRKtide, R14 makes hydrogen bonds with ATP as well as D2017 (DGY loop) of LRRK2. The structural equivalent position in PLK-peptide is occupied by L6 and does not participate in hydrogen bonding with ATP or D2017. This suggests that LRRKtide is likely to be more sensitive to mutations in the DYG-loop region compared to PLK-peptide. In the bottom-left panel, the interaction of LRRKtide^S with the active site of LRRK2 is shown in details. S12 of LRRKtide^S is in close proximity of charged residues including D2017 and D1994. In the bottom right-panel, the interaction of LRRKtide (contains Thr instead of Ser) with the active site of LRRK2 is shown in detail. The methyl group of T12 is placed between the oxygen of T12 and D1994 leading to charge shielding in this region.

Figure 2

Isotope exchange analysis for the mutant t-G2019S-catalyzed LRRKtide phosphorylation. Effect of [ADP]/[ATP] (A), [PO₄-LRRKtide]/[LRRKtide] (B), [ADP]/[LRRKtide] (C), and [PO₄-LRRKtide]/[ATP] (D) concentrations on the initial rates of the ATP to PO₄-LRRKtide isotopic exchanges. The concentrations of the varied reactants were maintained at a constant ratio of 20 while the other reactants were kept as 1 and 20 µM for the substrate and product, respectively. The trace amount of radioactive ATP was added prior to the initiation of the reaction by the addition of enzyme. The data in panel A and B were fit to the simple Michaelis-Menton equation, and data in panel C and D were fit to the equation reflecting the substrate inhibition: v = v_maxS/(K_m + [S](1 + [S]/K_i)).

Figure 3

Proton inventory and pL-dependent studies for t-wt LRRK2-catalyzed LRRKtide phosphorylaiton. For the proton inventory study, the initial velocities were measured at saturating concentrations of both ATP and peptide substrates for k_cat in the mixture of H₂O and D₂O at pH 8.2 and pD equivalent. The dependence of the ratio of k_cat (k_n/k₀) in the presence and absence of varying atom fractions of D₂O (n) on n revealed a normal SKIE of 1.1 (A). pL-dependent studies were carried out using a triple buffer consisting of 50 mM MES, 100 mM Tris, and 50 mM acetic acid. Panel B revealed pH (●) and pD (○) dependencies of k_cat with a SKIE of 1.1 and panel C revealed pH (●) and pD (○) dependencies of k_cat/K_m with a SKIE of 1.2.

Figure 4

Proton inventory and pL-dependent studies for t-wt LRRK2-catalyzed LRRKtide^S phosphorylation. Panel A shows proton inventory studies of LRRKtide^S phosphorylation. The inset shows the reciprocal of the ratio of k_cat (k₀/k_n) dependence on n. Panel B reveals pH (●) and pD (○) dependencies of k_cat with a SKIE of 0.65 and panel C reveals pH (●) and pD (○) dependencies of k_cat/K_m with a SKIE of 0.57.

Figure 5

LRRK2-catalyzed GTP hydrolysis. Initial velocities were measured as a function of [GTP] for t-wt LRRK2 (○) and the mutant t-G2019S (●).