Fluorescent BODIPY-GTP Analogs: Real-Time Measurement of Nucleotide Binding to G Proteins

McEwen, Dyke P.; Gee, Kyle R.; Kang, Hee Young; Neubig, Richard R.

doi:10.1006/abio.2001.5011

Cited by 127 publications

(127 citation statements)

References 25 publications

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“…This results in slow dissociation of the enzyme-product complex [EP] and renders the K m unrepresentative of the K d for GTP. To estimate the affinity of Roc ext for GTP, we determined the K d of Roc ext for GTP nonhydrolyzable analog GTPγS using a fluorescence polarization (FP) assay (16,17). As shown in Fig.…”

Section: Significancementioning

confidence: 99%

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

Liao

Burlak

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

105

136

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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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Section: Significancementioning

confidence: 99%

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

Liao

Burlak

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

105

136

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“…We also studied two other fluorescent nucleotides, BODIPY-FL-GTP␥S (compound 19) and BODIPY-FL-GMPPNP (compound 14), because those nucleotides possess more favorable optical properties than MANT-nucleotides (68). However, the BODIPY group is much bulkier than the MANT group, which property can cause steric problems in the binding of BODIPYnucleotides to target proteins (68,69).…”

Section: Potent and Selective Ac Inhibition By Mant-itp␥s Relative Tomentioning

confidence: 99%

“…However, the BODIPY group is much bulkier than the MANT group, which property can cause steric problems in the binding of BODIPYnucleotides to target proteins (68,69). The BODIPY group in compound 14 is attached to the 2Ј(3Ј)-O-position and undergoes spontaneous isomerization as does the MANT group in compounds 1-6, 8, 10, 11, and 16, whereas in compound 19, the BODIPY group is attached to the ␥-thiophosphate (68,69). We also examined the phosphorothioates ITP␥S (compound 13) and UTP␥S (compound 17) that are mixed G-protein activators and AC inhibitors (Fig.…”

Section: Potent and Selective Ac Inhibition By Mant-itp␥s Relative Tomentioning

confidence: 99%

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

Gille

Lushington

Mou

et al. 2004

Journal of Biological Chemistry

215

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ACs 1 catalyze the conversion of ATP into the second messenger cAMP, PP i being the second product of the cyclase reaction. Mammals express nine membranous ACs (ACs 1-9) (1, 2) and a sAC that is predominantly expressed in testis (3). Bacillus anthracis and Bacillus pertussis produce the AC toxins EF and ACT, respectively, that are activated by Ca 2ϩ /calmodulin and act through excessive cAMP accumulation in host cells (4,5). sGC is structurally related to ACs 1-9 in the catalytic site and is activated by [6][7][8]. sGC catalyzes the formation of the second messenger cGMP from GTP. ACs 1-9 contain a tandem repeat structure with two transmembrane domains and two cytosolic domains (1, 2). The cytosolic domains are referred to as C1 and C2, respectively. Together, C1 and C2 form the catalytic site of AC. C1 and C2 also contain the regulatory sites for the stimulatory G-protein, G␣ s , for the inhibitory G-protein, G␣ i , and for the diterpene, forskolin. Catalytic activity of all AC isoforms depends on the presence of divalent cations (Mg 2ϩ or Mn 2ϩ ). Membranous ACs possess two Me 2ϩ -binding sites (9 -11). When mixed together, purified C1 and C2 form a functional AC that is efficiently activated by forskolin and G␣ s -GTP␥S (12, 13).AC isoforms differ from each other in their regulation (1, 2). ACs 1-9 are all activated by G␣ s , whereas sAC is activated by HCO 3 Ϫ (14). Forskolin activates ACs 1-8 but not AC9 or sAC. G␣ i inhibits ACs 1, 5, and 6. G-protein ␤␥ subunits exhibit stimulatory or inhibitory effects on AC isoforms. Ca 2ϩ /calmodulin stimulates ACs 1, 3, and 8. In addition, Mg 2ϩ and Mn 2ϩ show differential stimulatory effects on AC isoforms (15). More-

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“…This results from the specific association of the G protein with GTPγS and the movement of the guanine base from the BODIPY group [28]. We therefore performed the fluorescence-based, real time GTPγS binding assay to verify Goα protein activity.…”

Section: Bodipy-gtp Analogs As a Tool To Measure Real-time Nucleotidementioning

confidence: 99%

Structural characterization of recombinant bovine Goαby spectroscopy and homology modeling

et al. 2011

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Abstract. Go, a member of heterotrimeric guanine nucleotide-binding proteins, is the most abundant form of G protein in the central and peripheral nervous systems. Goα has a significant role in neuronal development and function but its signal transduction mechanism remains to be clarified.In this study, the bovine Goα subunit was overexpressed and purified into homogeneity. Its activity was studied using [ 35 S] GTPγS binding, intrinsic fluorescence and BODIPY assays. The secondary structure was determined by both FTIR and CD spectroscopy as 42.3% α-helix, 13.4% β-sheet and 24.3% β-turn. A theoretical structure model was constructed. The structure from homology modeling is in very good agreement with the crystal structure of mouse Goα subunit except for the loop between αB-αC helices. This model was docked to the mouse RGS16 molecule. T117 on the αB-αC loop of Goα interacted with K172 on RGS16 as opposed to the T117 and K164 interaction in mouse.

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Fluorescent BODIPY-GTP Analogs: Real-Time Measurement of Nucleotide Binding to G Proteins

Cited by 127 publications

References 25 publications

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

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

Differential Inhibition of Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by Purine and Pyrimidine Nucleotides

Structural characterization of recombinant bovine Goαby spectroscopy and homology modeling

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