Structural Basis for the Inhibition of Mammalian Membrane Adenylyl Cyclase by 2 ′(3′)-O-(N-Methylanthraniloyl)-guanosine 5 ′-Triphosphate (original) (raw)
Related papers
Molecular …, 2011
2Ј,3Ј-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K i , 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329: 1156 -1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K i , 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket.
Journal of Biological Chemistry, 1995
Changes in conformation of the ⑀-subunit of the bovine heart mitochondrial F 1 -ATPase complex as a result of nucleotide binding have been demonstrated from the phosphorescence emission of tryptophan. The triplet state lifetime shows that whereas nucleoside triphosphate binding to the enzyme in the presence of Mg 2؉ increases the flexibility of the protein structure surrounding the chromophore, nucleoside diphosphate acts in an opposite manner, enhancing the rigidity of this region of the macromolecule. Such changes in dynamic structure of the ⑀-subunit are evident at high ligand concentration added to both the nucleotide-depleted F 1 (Nd-F 1 ) and the F 1 preparation containing the three tightly bound nucleotides ). Since the effects observed are similar in both the F 1 forms, the binding to the low affinity sites must be responsible for the conformational changes induced in the ⑀-subunit. This is partially supported by the observation that the Trp lifetime is not significantly affected by adding an equimolar concentration of adenine nucleotide to Nd-F 1 . The effects on protein structure of nucleotide binding to either catalytic or noncatalytic sites have been distinguished by studying the phosphorescence emission of the F 1 complex prepared with the three noncatalytic sites filled and the three catalytic sites vacant ). Phosphorescence lifetime measurements on this F 1 form demonstrate that the binding of Mg-NTP to catalytic sites induces a slight enhancement of the rigidity of the ⑀-subunit. This implies that the binding to the vacant noncatalytic site of F 1 (2,1) must exert the opposite and larger effect of enhancing the flexibility of the protein structure observed in both Nd-F 1 and F 1 (2,1). The observation that enhanced flexibility of the protein occurs upon addition of adenine nucleotides to F 1 (2,1) in the absence of Mg 2؉ provides direct support for this suggestion. The connection between changes in structure and the possible functional role of the ⑀-subunit is discussed.
Structural and energetic analysis of activation by a cyclic nucleotide binding domain
Journal of molecular …, 2008
MlotiK1 is a prokaryotic homolog of cyclic nucleotide-dependent ion channels which contains an intracellular C-terminal cyclic nucleotide binding domain (CNB domain). X-ray structures have been solved of the CNB domain in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding. We have used X-ray crystallography to determine three new MlotiK1 CNB domain structures: a second apo configuration, a cGMP-bound structure, and a second cAMP-bound structure. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo and bound states and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand-dependence in MlotiK1 and more globally in the class of nucleotide-dependent proteins.
Conformational Changes in the Catalytically Inactive Nucleotide Binding Site of CFTR
Biophysical Journal, 2013
A central step in the gating of the CFTR chloride channel is the association of its two cytosolic nucleotide binding domains (NBDs) into a head-to-tail dimer, with two nucleotides bound at the interface. Channel opening and closing, respectively, are coupled to formation and disruption of this tight NBD dimer. CFTR is an asymmetric ABC protein in which the two interfacial binding sites (composite sites 1 and 2) are functionally different. During gating the canonical, catalytically active, nucleotide binding site (site 2) cycles between dimerized prehydrolytic (state O 1 ), dimerized posthydrolytic (state O 2 ), and dissociated (state C) forms in a preferential C→O 1 →O 2 →C sequence. In contrast, the catalytically inactive nucleotide binding site (site 1) is believed to remain associated, ATPbound, for several gating cycles. Here we have examined the possibility of conformational changes in site 1 during gating, by studying gating effects of perturbations in site 1.
Bioorganic & Medicinal Chemistry Letters, 2010
a b s t r a c t (2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methylamino)benzamido]tetrahydrofuran-2-yl -methoxy[(hydroxy)phosphoryloxy][(hydroxy)phosphoryl]dichloromethylphosphonic acid was synthesized as a chemically and metabolically stable analog of ATP substituted with a fluorescent methylanthranoyl (MANT) residue. The compound is intended for studying the binding site and function of adenylyl cyclases (ACs), which was exemplified by studying its interaction with Bacillus anthracis edema factor (EF) AC exotoxin.
Functional Characterization of Two Nucleotide-binding Sites in Soluble Guanylate Cyclase
Journal of Biological Chemistry, 2006
Soluble guanylate cyclase is a heterodimeric hemoprotein composed of ␣and -subunits with a homologous motif to the nucleotide-binding sites of adenylate cyclases. Homology modeling of guanylate cyclase, based on the crystal structure of adenylate cyclase, reveals a single GTP-binding site and a putative second site pseudosymmetric to the GTP-binding site. However, the role of this pseudosymmetric site has remained unclear. Using equilibrium dialysis, we identified two nucleotide-binding sites with high and low affinity for ␣,-methylene guanosine 5-triphosphate (GMP-CPP). In contrast, 2-dADP occupied both sites with equivalent affinities. Adenosine-5-,␥-imido triphosphate (AMP-PNP), which competitively inhibited the cyclase reaction, bound solely to the high affinity site, indicating the role of this site as the catalytic site. The function of the low affinity site was examined using allosteric activators YC-1 and BAY 41-2272. YC-1 significantly reduced the affinity of 2-dADP, probably by competing for the same site as 2-dADP. BAY 41-2272 totally inhibited the specific binding of one molecule of 2-dADP as well as GMP-CPP. This suggests that the activators compete with these nucleotides for the low affinity site. Infrared and EPR analyses of the enzymic CO-and NO-hemes also supported the suggested role of the low affinity site as a target for the activators. Our results imply that the low affinity site is the pseudosymmetric site, which binds YC-1 or BAY 41-2272.
Journal of Biological Chemistry, 2003
2(3)-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5-[␥-thio]triphosphate (MANT-GTP␥S) and MANT-guanosine 5-[,␥imido]triphosphate (MANT-GppNHp) on G␣ sand G␣ iprotein-mediated signaling. MANT-GTP␥S/MANT-GppNHp had lower affinities for G␣ s and G␣ i than GTP␥S/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-G␣ fusion proteins. MANT-GTP␥S was much less effective than GTP␥S at disrupting the ternary complex between the formyl peptide receptor and G␣ i2. MANT-GTP␥S/MANT-GppNHp non-competitively inhibited GTP␥S/GppNHp-, AlF 4 ؊-,  2-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolinstimulated adenylyl cyclase (AC) in G␣ s-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTP␥S/MANT-GppNHp was not due to G␣ s inhibition because it was also observed in G␣ s-deficient S49 cyc ؊ lymphoma cell membranes. Mn 2؉ blocked AC inhibition by GTP␥S/ GppNHp in S49 cyc ؊ membranes but enhanced the potency of MANT-GTP␥S/MANT-GppNHp at inhibiting AC by ϳ4-8-fold. MANT-GTP␥S and MANT-GppNHp competitively inhibited forskolin/Mn 2؉-stimulated AC in S49 cyc ؊ membranes with K i values of 53 and 160 nM, respectively. The K i value for MANT-GppNHp at insect cell AC was 155 nM. Collectively, MANT-GTP␥S/MANT-GppNHp bind to G␣ sand G␣ i-proteins with low affinity and are ineffective at activating G␣. Instead, MANT-GTP␥S/ MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.
Journal of Pharmacology and Experimental Therapeutics, 2009
Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mou et al. (2006) Mol Pharmacol 70:878-86) we reported that 2',3'-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5'triphosphates (TNP-NTPs) are potent inhibitors (K i values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC) and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K i , 3.7 nM) and sGC (K i , 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K i , 7.1 nM) and ACII (K i , 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G sand G iproteins only with low potencies. Molecular modelling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and TNP-NTPs are a promising starting point to develop isoform-selective AC-and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.
Drug Metabolism and Disposition, 2007
Membranous adenylyl cyclases (ACs) play a key role in signal transduction and are promising drug targets. In previous studies we showed that 2',3'-(O)-(N-methylanthraniloyl) (MANT)substituted nucleotides are potent AC inhibitors. The aim of this study was to provide systematic structure-activity relationships for 21 (M)ANT-substituted nucleotides at the purified catalytic AC subunit heterodimer VC1:IIC2, the VC1:VC1 homodimer and recombinant ACs 1, 2 and 5. (M)ANT-nucleotides inhibited fully activated VC1:IIC2 in the order of affinity for bases hypoxanthine > uracil > cytosine > adenine ~ guanine ≫ xanthine. Omission of a hydroxyl group at the 2' or 3'-position reduced inhibitor potency as did introduction of a γ-thiophosphate group or omission of the γ-phosphate group. Substitution of the MANT-group by an ANT-group had little effect on affinity. Although all nucleotides bound to VC1:IIC2 similarly according to the tripartite pharmacophore model with a site for the base, the ribose, and the phosphate chain, nucleotides exhibited subtle differences in their binding modes as revealed by fluorescence spectroscopy and molecular modelling. MANT-nucleotides also differentially interacted with the VC1:VC1 homodimer as assessed by fluorescence spectroscopy and modelling. Similar structure-activity relationships as for VC1:IIC2 were obtained for recombinant ACs 1, 2 and 5, with AC2 being the least sensitive AC isoform in terms of inhibition. Overall, ACs possess a broad base-specificity with no preference for the "cognate" base adenine as verified by enzyme inhibition, fluorescence spectroscopy and molecular modelling. These properties of ACs are indicative for ligand-specific conformational landscapes that extend to the VC1:VC1 homodimer and should facilitate development of non-nucleotide inhibitors.