Parkinson disease-associated mutation R1441H in LRRK2 prolongs the “active state” of its GTPase domain

Abstract

Mutation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD). A disease-causing point mutation R1441H/G/C in the GTPase domain of LRRK2 leads to overactivation of its kinase domain. However, the mechanism by which this mutation alters the normal function of its GTPase domain [Ras of complex proteins (Roc)] remains unclear. Here, we report the effects of R1441H mutation (Roc R1441H ) on the structure and activity of Roc. We show that Roc forms a stable monomeric conformation in solution that is catalytically active, thus demonstrating that LRRK2 is a bona fide self-contained GTPase. We further show that the R1441H mutation causes a twofold reduction in GTPase activity without affecting the structure, thermal stability, and GDP-binding affinity of Roc. However, the mutation causes a twofold increase in GTP-binding affinity of Roc, thus suggesting that the PD-causing mutation R1441H traps Roc in a more persistently activated state by increasing its affinity for GTP and, at the same time, compromising its GTP hydrolysis.M utation in leucine-rich-repeat kinase 2 (LRRK2) is a common cause of Parkinson disease (PD) (1-5). LRRK2 is a large (2,527-aa) multidomain protein consisting of seven putative domains (2), including a Ras-like GTPase domain called Ras of complex proteins (Roc), followed by a domain called C-terminal of Roc (COR), which is then followed by a kinase domain (Kin). It remains unclear how perturbations of these activities result in disease; however, the most common mutation in LRRK2-associated PD, G2019S in the kinase domain, shows higher kinase activity than wild type; therefore, its overactivation might be associated with disease pathogenesis (6).The tandem Roc-COR-Kin arrangement suggests that their activities might be coupled such that the GTPase activity of Roc might modulate the kinase activity. Indeed, several studies have shown that GTP binding to the Roc domain regulates the activity of the Kin domain (7,8). Moreover, a PD-associated mutation in the Roc domain (R1441C) has been shown to have higher kinase activity (9), thus suggesting that mutations in the Roc domain, also up-regulate kinase activity.Understanding the function of Roc and its mechanism of action is important for understanding the mechanism of PD pathogenesis and therapeutic development. However, because of the lack sufficient quantity of protein samples amendable for detailed investigations, the biochemical properties and enzymatic activities of the Roc domain of LRRK2 are poorly understood.Here, we describe a stably folded construct of human Roc domain that enabled us to investigate quantitatively its biochemical and enzymatic properties. The results revealed that a PD-causing mutation R1441H in the Roc domain renders it less active at hydrolyzing GTP, as well as having higher affinity for GTP, than its wild-type counterpart, thereby increasing the residence time of its GTP-bound "active state," which is associated with PD pathogenesis (8). Results and DiscussionConstruction of a Stable Human LRR...

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Heterogeneity of Leucine-Rich Repeat Kinase 2 Mutations: Genetics, Mechanisms and Therapeutic Implications

“…The R1441 mutations cause a decrease in GTPase activity of LRRK2, although this activity is low and has been difficult to measure reliably, leading to some uncertainty about relevance of this observation [18][19][20]88]. The mechanism of diminished activity is likely via disruption of hydrogen bonding and stacking interactions normally provided by the arginine side chain [18][19][20]87], which may cause an enhancement in the affinity of LRRK2 for GTP with a subsequent decrease in GTP hydrolysis [89]. Substitution of the positively charged arginine to neutral amino acids (cytosine or glycine) is expected to cause misfolding of ROC domain [90].…”

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“…G2019S or I2020T), activate, or otherwise enhance, the proportion of autophosphorylated LRRK2 (6 -8). However, apart from the G2019S mutation that seems to enhance all aspects of kinase activity, other pathogenic mutations have variable effects on kinase activity, for example the R1441C and I2020T mutations that enhance the proportion of enzyme in an active state without affecting other kinetic parameters (9,10).…”