Inorganic phosphate binds to the empty nucleotide binding pocket of conventional myosin II

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times of Cy3-EDA-ATP (2'(3')-O-[[2-[[6-[2-[3-(1-ethyl-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-5-sulfo-3H-indolio]-1-oxohexyl]amino]ethyl]carbamoyl]ATP) but reduced the number of Cy3-signals per field of view, approaching 50% at phosphate concentrations of 1-2 mM. Apparently, inhibition of movement does not result from binding of phosphate to an actomyosin-ADP intermediate as proposed by Hooft and coworkers (Hooft, A. M., Maki, E. J., Cox, K. K., and Baker, J. E. (2007) Biochemistry 46, 3513-3520) but, rather, from forming a strong-binding actomyosin-phosphate intermediate.     

A competitive nucleotide binding inhibitor: in vitro characterization of Rab7 GTPase inhibition

Mapping the functionality of GTPases through small molecule inhibitors represents an underexplored area in large part due to the lack of suitable compounds. Here we report on the small chemical molecule 2-(benzoylcarbamothioylamino)-5,5-dimethyl-4,7-dihydrothieno[2,3-c]pyran-3-carboxylic acid (PubChem CID 1067700) as an inhibitor of nucleotide binding by Ras-related GTPases. The mechanism of action of this pan-GTPase inhibitor was characterized in the context of the Rab7 GTPase as there are no known inhibitors of Rab GTPases. Bead-based flow cytometry established that CID 1067700 has significant inhibitory potency on Rab7 nucleotide binding with nanomolar inhibitor (K(i)) values and an inhibitory response of ≥97% for BODIPY-GTP and BODIPY-GDP binding. Other tested GTPases exhibited significantly lower responses. The compound behaves as a competitive inhibitor of Rab7 nucleotide binding based on both equilibrium binding and dissociation assays. Molecular docking analyses are compatible with CID 1067700 fitting into the nucleotide binding pocket of the GTP-conformer of Rab7. On the GDP-conformer, the molecule has greater solvent exposure and significantly less protein interaction relative to GDP, offering a molecular rationale for the experimental results. Structural features pertinent to CID 1067700 inhibitory activity have been identified through initial structure-activity analyses and identified a molecular scaffold that may serve in the generation of more selective probes for Rab7 and other GTPases. Taken together, our study has identified the first competitive GTPase inhibitor and demonstrated the potential utility of the compound for dissecting the enzymology of the Rab7 GTPase, as well as serving as a model for other small molecular weight GTPase inhibitors.     

The interaction of Phe472 with a fluorescent inhibitor bound to the complex of myosin subfragment-1 with nucleotide

The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA) acts as a fluorescent inhibitor for the ATPases of skeletal [Hiratsuka (1994) J. Biol. Chem. 269, 27251-27257] and Dictyostelium discoideum [Bobkov et al. (1997) J. Muscle Res. Cell Motil. 18, 563-571] myosins. The former paper suggested that, upon addition of excess nucleotides to the binary complex of subfragment-1 from skeletal myosin (S1) with PPBA, a stable ternary complex of S1 with PPBA and nucleotide is formed. Useful fluorescence properties of PPBA enable us to distinguish the conformation of the myosin ATPase at the ATP state from that at the ADP state. In the present paper, to determine the PPBA-binding site in the complexes, enzymatic and fluorescence properties of the S1.PPBA.nucleotide complexes were investigated. Upon formation of the ternary complex with ATP, a new peak appeared at 398 nm in the PPBA fluorescence spectrum. Experiments using model compounds of aromatic amino acid suggested that this fluorescence peak at 398 nm is originated from PPBA interacting with Phe residue(s). Taking into account differences in fluorescence spectra between complexes of S1 and those of subfragment-1 from D. discoideum myosin (S1dC), in the ternary complex of S1 formed with ATP, PPBA was suggested to interact with Phe residue(s) that is absent in S1dC. Docking simulation of PPBA on the S1.nucleotide complex revealed that Phe472 interacts with PPBA. Binding sites of PPBA and blebbistatin, an inhibitor showing high affinity and selectivity toward myosin II [Kovács et al. (2004) J. Biol. Chem. 279, 35557-35563], seem to overlap at least partly.     

Flightin is a myosin rod binding protein

The assembly of striated muscle myosin into thick filaments of precise and regular length requires the assistance of accessory proteins. Drosophila indirect flight muscle (IFM) contain flightin, a 20-kDa protein that has been shown to be essential for flight, for maintenance of sarcomeric integrity in active muscle, and informative in length determination of thick filaments during IFM development. Additionally, a point mutation in the myosin rod (Mhc ¹³) negates flightin accumulation in the IFM in vivo. The manner in which flightin interacts with thick filaments is not known. Here, two different solid-state binding assays demonstrate that flightin binds to myosin and to a recombinant fragment of the myosin rod that include the COOH-terminal 600 amino acids (zone 19 to tail piece). The interaction of flightin and myosin is abolished by the single amino acid substitution in Mhc ¹³ at position 1e of zone 27 of the red (residue 1554). The molar ratio of flightin to myosin is approx 1∶1 to 1∶2. Thus, the instability of thick filaments, seen in vivo in the absence of flightin suggests that the flightin-myosin interaction is critical for maintaining sarcomere integrity in active muscle.     

Interactions of the actin and nucleotide binding sites on myosin subfragment 1.

The effects of selected nucleotides (N) on the binding of myosin subfragment 1 (S-1) and pure F-actin (A) were measured by time-resolved fluorescence depolarization for 0.15 M KCl, pH 7.0 at 4 degrees. The association constants K'A, KN, and K'N in the scheme (see article), were determined for the magnesium salts of ADP, adenyl-5'-yl imidodiphosphate AMP-P(NH)P, and PPi. The nucleotide binding site on S-1 was "mapped" with respect to its interaction on the actin binding site. The subsites were the beta- and gamma-phosphoryl groups of ATP bind had the largest effects. A quantitative measure of the interaction, the interaction free energy, was defined as -RT ln (KA/K'A). For ADP, K'A was 2.7 X 10(5) M-1 and the interaction free energy was -4.67 kJ M-1. For AMP-P(NH)P and PPi it was much larger. A ternary complex was shown to exist for ADP, S-1, and actin in the presence of Mg2+ and evidence from AMP-P(NH)P and PPi measurements indicated that ATP also likely forms a ternary complex. The mechanism of (S-1)-actin dissociation is discussed in light of these results.     

Lauroylethanolamide is a potent competitive inhibitor of lipoxygenase activity

The lipoxygenase (LOX) pathway was proposed to compete with hydrolysis and be partly responsible for the metabolism of polyunsaturated N-acylethanolamines (PU-NAEs). Treatment of Arabidopsis seedlings with lauroylethanolamide (NAE 12:0) resulted in elevated levels of PU-NAE species, and this was most pronounced in plants with reduced NAE hydrolase activity. Enzyme activity assays revealed that NAE 12:0 inhibited LOX-mediated oxidation of PU lipid substrates in a dose-dependent and competitive manner. NAE 12:0 was 10-20 times more potent an inhibitor of LOX activities than lauric acid (FFA 12:0). Furthermore, treatment of intact Arabidopsis seedlings with NAE 12:0 (but not FFA 12:0) substantially blocked the wound-induced formation of jasmonic acid (JA), suggesting that NAE 12:0 may be used in planta to manipulate oxylipin metabolism.     

Enthalpy of nucleotides binding to myosin.

The enthalpies of binding adenosine 5'-diphosphate (ADP) and 5'-adenylyl imidodiphosphate [AMP-P(NH)P] to rabbit skeletal myosin have been measured in Pipes and Tris buffers at pH 7.8 and 15 degrees C. For ADP the enthalpy of binding was exothermic, whereas the enthalpy of binding AMP-P(NH)P, a nonhydrolyzable ATP analogue, was small and endothermic. For the reaction of ATP and myosin, the development of enthalpy was resolved into two phases: a fast endothermic phase, which is the summation of binding and hydrolysis, and a slow exothermic phase, which is associated with product-release steps. These results are discussed in terms of their implications for energy transduction.     

Kinetic properties of binding of myosin subfragment-1 with F-actin in the absence of nucleotide

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.     

Analysis of nucleotide binding to Dictyostelium myosin II motor domains containing a single tryptophan near the active site

Dictyostelium myosin II motor domain constructs containing a single tryptophan residue near the active sites were prepared in order to characterize the process of nucleotide binding. Tryptophan was introduced at positions 113 and 131, which correspond to those naturally present in vertebrate skeletal muscle myosin, as well as position 129 that is also close to the adenine binding site. Nucleotide (ATP and ADP) binding was accompanied by a large quench in protein fluorescence in the case of the tryptophans at 129 and 131 but a small enhancement for that at 113. None of these residues was sensitive to the subsequent open-closed transition that is coupled to hydrolysis (i.e. ADP and ATP induced similar fluorescence changes). The kinetics of the fluorescence change with the F129W mutant revealed at least a three-step nucleotide binding mechanism, together with formation of a weakly competitive off-line intermediate that may represent a nonproductive mode of nucleotide binding. Overall, we conclude that the local and global conformational changes in myosin IIs induced by nucleotide binding are similar in myosins from different species, but the sign and magnitude of the tryptophan fluorescence changes reflect nonconserved residues in the immediate vicinity of each tryptophan. The nucleotide binding process is at least three-step, involving conformational changes that are quite distinct from the open-closed transition sensed by the tryptophan Trp(501) in the relay loop.     

Fumaric acid is a competitive inhibitor of wheat germ lipoxygenase

Fumaric acid is shown to be a competitive inhibitor of wheat germ lipoxygenase.     

A functional analysis of the allosteric nucleotide monophosphate binding site of carbamoyl phosphate synthetase

The catalytic activity of carbamoyl phosphate synthetase (CPS) from Escherichia coli is allosterically regulated by UMP, IMP, and ornithine. Thirteen amino acids within the domain that harbors the overlapping binding sites for IMP and UMP were mutated to alanine and characterized. The four residues that interact directly with the phosphate moiety of IMP in the X-ray crystal structure (K954, T974, T977, and K993) were shown to have the greatest impact on the dissociation constants for the binding of IMP and UMP and the associated allosteric effects on the kinetic constants of CPS. Of the four residues that interact with the ribose moiety of IMP (S948, N1015, T1017, and S1026), S1026 was shown to be more important for the binding of IMP than UMP. Five residues (V994, I1001, D1025, V1028, and I1029) were mutated in the region of the allosteric domain that surrounds the hypoxanthine ring of IMP. With the exception of V994A, these mutations had a modest influence on the binding and subsequent allosteric effects by UMP and IMP.     

Acarbose is a competitive inhibitor of mammalian lysosomal acid alpha-D-glucosidases

Intraperitoneal injections (approximately 400 mg/kg of body weight) of acarbose, an inhibitor of acid (1----4)-alpha-D-glucosidase, perturb the metabolism of glycogen in the liver, resulting in excess storage of lysosomal glycogen. The metabolism of skeletal muscle glycogen was unaffected, suggesting that acarbose either does not enter the tissue or that the muscle alpha-D-glucosidase is not inhibited. The hydrolysis of maltose and glycogen by the acid alpha-D-glucosidases from rat liver, rat skeletal muscle, and human placenta was inhibited competitively by acarbose. Thus, the lack of effect of acarbose upon the metabolism of muscle glycogen is due to its inability to enter the tissue.     

Pyrazinamide, but not pyrazinoic acid, is a competitive inhibitor of NADPH binding to Mycobacterium tuberculosis fatty acid synthase I

Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty acid synthase I (FAS I). Both PZA and pyrazinoic acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.     

A study of the binding of adenosine diphosphate to myosin subfragment-1.

The binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.     

D-dihexanoylphosphatidylcholine is not a pure competitive inhibitor of phospholipase A2 hydrolysis of L-dihexanoylphosphatidylcholine

The inhibition of Crotalus adamanteus phospholipase A2 hydrolysis of L-dihexanoylphosphatidylcholine by D-dihexanoylphosphatidylcholine was investigated with inhibitor and substrate in the monomeric concentration range. The results showed that the D-enantiomer acts as a partial (not pure) competitive inhibitor of the enzyme. These results suggest that an ESI complex exists, in which hydrolysis of the substrate still occurs. Thus, binding of the D-enantiomer to the enzyme decreases the affinity for the substrate by a factor, alpha, while Vmax is unaffected. The value of alpha was determined to be 4.70 +/- 0.14. These findings complicate the use of D-phosphatidylcholines in mixed micelles with the L-enantiomer as a possible method to investigate the mechanism of interfacial activation of this enzyme.     

Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.     

Interaction of F-actin-AMPPNP with myosin subfragment-1 and troponin-tropomyosin: influence of an extra phosphate at the nucleotide binding site in F-actin on its function

An unsplitable analogue of ATP (adenylyl imidodiphosphate; AMPPNP) was incorporated into F-actin [Cooke, R. (1975) Biochemistry 14, 3250-3256]. The resulting polymers (F-actin-AMPPNP) activated the ATPase activity of myosin subfragment-1 (S1) as efficiently as normal F-actin; neither the maximum velocity at infinite actin concentration (Vmax) nor the affinity of actin to S1 in the presence of ATP (1/KATPase) changed, which indicates that the terminal phosphate of the bound nucleotide at the cleft region between the two domains of the actin molecule [Kabsch, W., Mannherz, H.G., & Suck, D. (1985) EMBO J. 4, 2113-2118] is not directly involved in a myosin binding site. However, the interaction of F-actin with troponin-tropomyosin was strongly modulated by the replacement of ADP with AMPPNP. The troponin-tropomyosin complex strongly enhanced the activation of S1-ATPase activity by F-actin-AMPPNP in the presence of Ca2+, although it has no effect on the activation by normal F-actin-ADP. KATPase was enhanced about threefold by troponin-tropomyosin in the presence of Ca2+, while Vmax was not markedly changed. F-actin-AMPPNP is highly potentiated by troponin-tropomyosin even with low S1 to actin ratios and at high ATP conditions. In the absence of Ca2+, the activation by F-actin-AMPPNP was inhibited normally by troponin-tropomyosin. The results suggest that the terminal beta-phosphate of the bound nucleotide in F-actin is located in a region which is important for regulation of the interaction with myosin.     

ADF/cofilin regulates actomyosin assembly through competitive inhibition of myosin II binding to F-actin

The contractile actin cortex is important for diverse fundamental cell processes, but little is known about how the assembly of F-actin and myosin II motors is regulated. We report that depletion of actin depolymerizing factor (ADF)/cofilin proteins in human cells causes increased contractile cortical actomyosin assembly. Remarkably, our data reveal that the major cellular defects resulting from ADF/cofilin depletion, including cortical F-actin accumulation, were largely due to excessive myosin II activity. We identify that ADF/cofilins from unicellular organisms to humans share a conserved activity to inhibit myosin II binding to F-actin, indicating a mechanistic rationale for our cellular results. Our study establishes an essential requirement for ADF/cofilin proteins in the control of normal cortical contractility and in processes such as mitotic karyokinesis. We propose that ADF/cofilin proteins are necessary for controlling actomyosin assembly and intracellular contractile force generation, a function of equal physiological importance to their established roles in mediating F-actin turnover.