The Parkinson disease-linked LRRK2 protein mutation I2020T stabilizes an active state conformation leading to increased kinase activity.
The effect of leucine-rich repeat kinase 2 (LRRK2) mutation I2020T on its kinase activity has been controversial, with both increased and decreased effects being reported. We conducted steady-state and pre-steady-state kinetic studies on LRRKtide and its analog LRRKtide(S). Their phosphorylation differs by the rate-limiting steps: product release is rate-limiting for LRRKtide and phosphoryl transfer is rate-limiting for LRRKtide(S). As a result, we observed that the I2020T mutant is more active than wild type (WT) LRRK2 for LRRKtide(S) phosphorylation, whereas it is less active than WT for LRRKtide phosphorylation. Our pre-steady-state kinetic data suggest that (i) the I2020T mutant accelerates the rates of phosphoryl transfer of both reactions by 3-7-fold; (ii) this increase is masked by a rate-limiting product release step for LRRKtide phosphorylation; and (iii) the observed lower activity of the mutant for LRRKtide phosphorylation is a consequence of its instability: the concentration of the active form of the mutant is 3-fold lower than WT. The I2020T mutant has a dramatically low KATP and therefore leads to resistance to ATP competitive inhibitors. Two well known DFG-out or type II inhibitors are also weaker toward the mutant because they inhibit the mutant in an unexpected ATP competitive mechanism. The I2020 residue lies next to the DYG motif of the activation loop of the LRRK2 kinase domain. Our modeling and metadynamic simulations suggest that the I2020T mutant stabilizes the DYG-in active conformation and creates an unusual allosteric pocket that can bind type II inhibitors but in an ATP competitive fashion.
From the Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, and.
Soumya
S
Ray
Samantha
S
Bender
Stephanie
S
Kang
Regina
R
Lin
Marcie A
MA
Glicksman
Min
M
Liu
D000255:Adenosine Triphosphate; D019943:Amino Acid Substitution; D000818:Animals; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008954:Models, Biological; D008958:Models, Molecular; D020125:Mutation, Missense; D010300:Parkinson Disease / Q000201:enzymology / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary
D000255:Adenosine Triphosphate; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases
Enzyme Inhibitors; Enzyme Kinetics; LRRK2; Molecular modeling; Parkinson Disease