MgATP Binding Research Articles

Pim Kinase Inhibitors Evaluated with a Single‐Molecule Engineered Nanopore Sensor

AbstractProtein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label‐free determination of inhibition constants. The procedure circumvents many sources of bias or false‐positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.

Pim Kinase Inhibitors Evaluated with a Single-Molecule Engineered Nanopore Sensor.

Protein kinases are critical therapeutic targets. Pim kinases are implicated in several leukaemias and cancers. Here, we exploit a protein nanopore sensor for Pim kinases that bears a pseudosubstrate peptide attached by an enhanced engineering approach. Analyte binding to the sensor peptide is measured through observation of the modulation of ionic current through a single nanopore. We observed synergistic binding of MgATP and kinase to the sensor, which was used to develop a superior method to evaluate Pim kinase inhibitors featuring label-free determination of inhibition constants. The procedure circumvents many sources of bias or false-positives inherent in current assays. For example, we identified a potent inhibitor missed by differential scanning fluorimetry. The approach is also amenable to implementation on high throughput chips.

Myosin MgADP Release Rate Decreases at Longer Sarcomere Length to Prolong Myosin Attachment in Skinned Rat Myocardial Strips

Cardiac contractility increases as sarcomere length (SL) increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. Myosin's capacity to generate force is Ca2+-regulated by thin-filament proteins and sarcomere length, which dictates the number of potential actin-myosin cross-bridge interactions. One mechanism underlying greater cardiac contractility at longer SL could involve longer myosin attachment duration (ton). To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm SL. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate (k-ADP) and MgATP binding rate (k+ATP). As MgATP increased ton decreased hyperbolically for both SL, but ton was roughly 50% longer for 2.2 vs. 1.9 µm SL at each [MgATP] (25±3 vs. 16±1 ms at 5 mM MgATP, 17° C, p<0.05). These ton differences arose from slower k-ADP at 2.2 µm SL (42±3 vs. 74±8 s−1, p<0.001), as MgATP binding rates did not differ with SL (281±56 vs. 327±93 mM−1 s−1). Absolute tension values were greater at 2.2 vs. 1.9 µm SL for relaxed (4.4±0.7 vs. 0.8±0.2 kPa at pCa 8.0, p<0.001) and maximally activated (20.0±1.4 vs. 14.2±1.6 kPa at pCa 4.8, p<0.001) conditions, and the force-pCa relationship was more sensitive to Ca2+ at 2.2 µm SL (pCa50=5.45±0.01 vs. 5.36±0.01, p<0.05). These increased tension values suggest that cross-bridges may bear greater loads at longer SL, which diminishes MgADP release to prolong ton and amplify cooperative cross-bridge contributions to thin filament activation. Therefore, load-dependent rates of the actomyosin cross-bridge cycle may vary with SL to contribute, in part, to the Frank-Starling relationship in the heart.

Sulfonylureas suppress the stimulatory action of Mg-nucleotides on Kir6.2/SUR1 but not Kir6.2/SUR2A KATP channels: a mechanistic study.

Sulfonylureas, which stimulate insulin secretion from pancreatic β-cells, are widely used to treat both type 2 diabetes and neonatal diabetes. These drugs mediate their effects by binding to the sulfonylurea receptor subunit (SUR) of the ATP-sensitive K(+) (KATP) channel and inducing channel closure. The mechanism of channel inhibition is unusually complex. First, sulfonylureas act as partial antagonists of channel activity, and second, their effect is modulated by MgADP. We analyzed the molecular basis of the interactions between the sulfonylurea gliclazide and Mg-nucleotides on β-cell and cardiac types of KATP channel (Kir6.2/SUR1 and Kir6.2/SUR2A, respectively) heterologously expressed in Xenopus laevis oocytes. The SUR2A-Y1206S mutation was used to confer gliclazide sensitivity on SUR2A. We found that both MgATP and MgADP increased gliclazide inhibition of Kir6.2/SUR1 channels and reduced inhibition of Kir6.2/SUR2A-Y1206S. The latter effect can be attributed to stabilization of the cardiac channel open state by Mg-nucleotides. Using a Kir6.2 mutation that renders the KATP channel insensitive to nucleotide inhibition (Kir6.2-G334D), we showed that gliclazide abolishes the stimulatory effects of MgADP and MgATP on β-cell KATP channels. Detailed analysis suggests that the drug both reduces nucleotide binding to SUR1 and impairs the efficacy with which nucleotide binding is translated into pore opening. Mutation of one (or both) of the Walker A lysines in the catalytic site of the nucleotide-binding domains of SUR1 may have a similar effect to gliclazide on MgADP binding and transduction, but it does not appear to impair MgATP binding. Our results have implications for the therapeutic use of sulfonylureas.

Kinetics of cardiac myosin isoforms in mouse myocardium are affected differently by presence of myosin binding protein-C.

We tested whether cardiac myosin binding protein-C (cMyBP-C) affects myosin cross-bridge kinetics in the two cardiac myosin heavy chain (MyHC) isoforms. Mice lacking cMyBP-C (t/t) and transgenic controls (WT(t/t)) were fed L-thyroxine (T4) to induce 90/10% expression of α/β-MyHC. Non-transgenic (NTG) and t/t mice were fed 6-n-propyl-2-thiouracil (PTU) to induce 100% expression of β-MyHC. Ca(2+)-activated, chemically-skinned myocardium underwent length perturbation analysis with varying [MgATP] to estimate the MgADP release rate (k(-ADP)) and MgATP binding rate (k(+ATP)). Values for (k(-ADP)) were not significantly different between t/t(T4) (102.2 ± 7.0 s(-1)) and WT(t/t)(T4) (91.3 ± 8.9 s(-1)), but k(+ATP)) was lower in t/t(T4) (165.9 ± 12.5 mM(-1) s(-1)) compared to WT(t/t)(T4) (298.6 ± 15.7 mM(-1) s(-1), P < 0.01). In myocardium expressing β-MyHC, values for k(-ADP) were higher in t/t(PTU) (24.8 ± 1.0 s(-1)) compared to NTG(PTU) (15.6 ± 1.3 s(-1), P < 0.01), and k(+ATP) was not different. At saturating [MgATP], myosin detachment rate approximates k(-ADP), and detachment rate decreased as sarcomere length (SL) was increased in both t/t(T4) and WT(t/t)(T4) with similar sensitivities to SL. In myocardium expressing β-MyHC, detachment rate decreased more as SL increased in t/t(PTU) (21.5 ± 1.3 s(-1) at 2.2 μm and 13.3 ± 0.9 s(-1) at 3.3 μm) compared to NTGPTU (15.8 ± 0.3 s(-1) at 2.2 μm and 10.9 ± 0.3 s(-1) at 3.3 μm) as detected by repeated-measures ANOVA (P < 0.01). These findings suggest that cMyBP-C reduces MgADP release rate for β-MyHC, but not for α-MyHC, even as the number of cMyBP-C that overlap with the thin filament is reduced to zero. Therefore, cMyBP-C appears to affect β-MyHC kinetics independent of its interaction with the thin filament.

Successful development and use of a thermodynamic stability screen for optimizing the yield of nucleotide binding domains

ATP sensitive potassium (KATP) channels consist of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B). SUR proteins are members of the ATP-binding cassette superfamily of proteins. Binding of ATP to the Kir6.x subunit mediates channel inhibition, whereas MgATP binding and hydrolysis at the SUR NBDs results in channel opening. Mutations in SUR1 and SUR2A NBDs cause diseases of insulin secretion and cardiac disorders, respectively, underlying the importance of studying the NBDs. Although purification of SUR2A NBD1 in a soluble form is possible, the lack of long-term sample stability of the protein in a concentrated form has precluded detailed studies of the protein aimed at gaining a molecular-level understanding of how SUR mutations cause disease. Here we use a convenient and cost-effective thermodynamic screening method to probe stabilizing conditions for SUR2A NBD1. Results from the screen are used to alter the purification protocol to allow for significantly increased yields of the purified protein. In addition, the screen provides strategies for long-term storage of NBD1 and generating NBD1 samples at high concentrations suitable for NMR studies. NMR spectra of NBD1 with MgAMP-PNP are of higher quality compared to using MgATP, indicating that MgAMP-PNP be used as the ligand in future NMR studies. The screen presented here can be expanded to using different additives and can be employed to enhance purification yields, sample life times, and storage of other low stability nucleotide binding domains, such as GTPases.

Functional CBS modules make IMPDHs octameric

Inosine-5'-monophosphate dehydrogenase (1, 2) (IMPDH) is a major target for antiviral, antiparasitic, antileukemic and immunosuppressive therapies. It is an ubiquitous and essential enzyme for the biosynthesis of guanosine nucleotides. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter had no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent indication of their importance in vivo. The aim of our study was to provide evidence for the role of the CBS modules on the quaternary structure and on the regulation of IMPDHs. A multidisciplinary approach involving enzymology, site-directed mutagenesis, analytical ultracentrifugation, X-ray crystallography, SAXS, cryo-electron microscopy and molecular modelling allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is functionally active as an octamer and allosterically regulated by MgATP via each CBS module. Revisiting deposited structural data, we found this newly discovered octameric organization conserved in other IMPDH structures. Meanwhile, we demonstrated that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N, localised into the CBS domains, appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Our data (3) revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules, inducing large structural rearrangements. Thus, the regulatory CBS modules in IMPDHs are functional and they can either modulate catalysis or/and macromolecular assembly. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop antibacterial compounds or drugs to counter retinopathy onset.

Coordinating Role of His216 in MgATP Binding and Cleavage in Pyruvate Carboxylase

His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3′-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305.

Non-Linear Cross-Bridge Elasticity, ATP-Independent Detachment and ATP-Velocity Relationships for Skeletal Muscle Actomyosin

The idea that contraction of skeletal muscle and heart results from ATP-driven actomyosin cross-bridge cycles is generally accepted. However, operational details remain controversial. For instance, in conflict with most accepted views, evidence was recently presented [1] for appreciably non-linear elasticity with low stiffness for post-power-stroke cross-bridges. Moreover, a non-hyperbolic relationship was observed [2] between MgATP concentration and sliding velocity for actin filaments propelled in vitro by myosin subfragment 1 or full length myosin. Here we present convincing evidence for a hyperbolic [MgATP]-velocity relationship (r2=0.998; Michaelis-Menten constants, Vmax=15.28 ± 0.28 µm/s (mean±SEM) and KM= 0.389 ± 0.023 mM) when actin filaments are propelled by heavy meromyosin from rabbit fast skeletal muscle myosin (28-29oC; >3 independent experiments). Because the hyperbolic [MgATP]-velocity relationship is not readily consistent with inter-head cooperativity the results were interpreted using a cross-bridge model with independent myosin heads. The inter-state transition rates were strain-dependent and the model had one detached state and five attached actomyosin (AM) states with either MgATP (AMATP) or MgADP and/or inorganic phosphate (Pi) or no nucleotide at the active site. The AMADPPi state was a strongly bound pre-power-stroke state whereas the remaining states without Pi were post-power-stroke states required to account for strain-dependent MgADP-release on the one hand and MgATP-dependence of velocity and competitive inhibition of MgATP binding by MgADP (AM, AMADP, AMATP) on the other. The MgATP induced detachment was supplemented by MgATP independent, but strain-dependent, detachment from the rigor (AM) state. This model predicts a hyperbolic [MgATP]-velocity relationship if the cross-bridge elasticity is non-linear but a non-hyperbolic [MgATP]-velocity relationship (cf. [2]) if cross-bridge elasticity is linear.

The Allosteric Role of the AAA+ Domain of ChlD Protein from the Magnesium Chelatase of Synechocystis Species PCC 6803

Magnesium chelatase is an AAA(+) ATPase that catalyzes the first step in chlorophyll biosynthesis, the energetically unfavorable insertion of a magnesium ion into a porphyrin ring. This enzyme contains two AAA(+) domains, one active in the ChlI protein and one inactive in the ChlD protein. Using a series of mutants in the AAA(+) domain of ChlD, we show that this site is essential for magnesium chelation and allosterically regulates Mg(2+) and MgATP(2-) binding.

Metal Switch-controlled Myosin II from Dictyostelium discoideum Supports Closure of Nucleotide Pocket during ATP Binding Coupled to Detachment from Actin Filaments

G-proteins, kinesins, and myosins are hydrolases that utilize a common protein fold and divalent metal cofactor (typically Mg(2+)) to coordinate purine nucleotide hydrolysis. The nucleoside triphosphorylase activities of these enzymes are activated through allosteric communication between the nucleotide-binding site and the activator/effector/polymer interface to convert the free energy of nucleotide hydrolysis into molecular switching (G-proteins) or force generation (kinesins and myosin). We have investigated the ATPase mechanisms of wild-type and the S237C mutant of non-muscle myosin II motor from Dictyostelium discoideum. The S237C substitution occurs in the conserved metal-interacting switch-1, and we show that this substitution modulates the actomyosin interaction based on the divalent metal present in solution. Surprisingly, S237C shows rapid basal steady-state Mg(2+)- or Mn(2+)-ATPase kinetics, but upon binding actin, its MgATPase is inhibited. This actin inhibition is relieved by Mn(2+), providing a direct and experimentally reversible linkage of switch-1 and the actin-binding cleft through the swapping of divalent metals in the reaction. Using pyrenyl-labeled F-actin, we demonstrate that acto·S237C undergoes slow and weak MgATP binding, which limits the rate of steady-state catalysis. Mn(2+) rescues this effect to near wild-type activity. 2'(3')-O-(N-Methylanthraniloyl)-ADP release experiments show the need for switch-1 interaction with the metal cofactor for tight ADP binding. Our results are consistent with strong reciprocal coupling of nucleoside triphosphate and F-actin binding and provide additional evidence for the allosteric communication pathway between the nucleotide-binding site and the filament-binding region.

ModulatoryATPbinding to the E2 state of maize plasma membrane H+‐ATPase indicated by the kinetics of vanadate inhibition

P-type ATPases, as major consumers of cellular ATP in eukaryotic cells, are characterized by the formation of a phosphorylated enzyme intermediate (E2P), a process that is allosterically coupled to translocation of cations against an electrochemical gradient. The catalytic cycle comprises binding of Mg-ATP at the nucleotide-binding domain, phosphorylation of the E1 state (E1), conformational transition to the E2P state, and dephosphorylation through the actuator domain and re-establishment of the E1 state. Recently, it has been suggested that, for several P-type ATPases, Mg-ATP binds to the phosphorylated enzyme, thereby accelerating the transition to the E1 state, before then becoming the enzyme's catalytic substrate. Here, we provide evidence supporting this viewpoint. We employed kinetic models based on steady-state kinetics in the presence and absence of the reversible inhibitor orthovanadate. Vanadate is generally considered to be a conformational probe that specifically binds to the E2 state, arresting the enzyme in a state analogous to the E2P state. Hydrolytic H(+) -ATPase activities were measured in inside-out plasma membrane vesicles isolated from roots and shoots of maize plants. For root enzymes, kinetic models of vanadate inhibition that allow simultaneous binding of Mg-ATP and vanadate to the same enzyme state were most plausible. For shoot enzymes, application of the competitive inhibitor Mg-free ATP attenuated vanadate inhibition, which is consistent with a model in which either Mg-free ATP or Mg-ATP is bound to the enzyme when vanadate binds. Therefore, data from roots and shoots indicate that binding of ATP species before transition to the E1 state plays an important role in the catalytic cycle of plant plasma membrane H(+) -ATPase.

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K(ATP) channels, (SUR1/Kir6.2)4, couple the transmembrane flux of K(+), and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5'-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR(1E1507Q), with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5'-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[(32)P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K(ATP) channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.

Protein-Mediated Antagonism between HIV Reverse Transcriptase Ligands Nevirapine and MgATP

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT.

Elevated rates of force development and MgATP binding in F764L and S532P myosin mutations causing dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a disease characterized by dilation of the ventricular chambers and reduced contractile function. We examined the contractile performance of chemically-skinned ventricular strips from two heterozygous murine models of DCM-causing missense mutations of myosin, F764L/+ and S532P/+, in an α-myosin heavy chain (MyHC) background. In Ca2+-activated skinned myocardial strips, the maximum developed tension in F764L/+ was only ~50% that of litter-mate controls (+/+). The F764L/+ also exhibited significantly reduced rigor stiffness, loaded shortening velocity and power output. Corresponding indices for S532P/+ strips were not different from controls. Manipulation of MgATP concentration in conjunction with measures of viscoelasticity, which provides estimates of myosin detachment rate 2πc, allowed us to probe the molecular basis of changes in crossbridge kinetics that occur with the myosin mutations. By examining the response of detachment rate to varying MgATP we found the rate of MgADP release was unaffected by the myosin mutations. However, MgATP binding rate was higher in the DCM groups compared to controls (422±109mM−1·s−1 in F764L/+, 483±74mM−1·s−1 in S532P/+ and 303±18mM−1·s−1 in +/+). In addition, the rate constant of force development, 2πb, was significantly higher in DCM groups compared to controls (at 5mM MgATP: 36.9±4.9s−1 in F764L/+, 32.9±4.5s−1 in S532P/+ and 18.2±1.7s−1 in +/+). These results suggest that elevated rates of force development and MgATP binding are features of cardiac myofilament function that underlie the development of DCM.

Cardiac Myosin Binding Protein-C Influences the Rates of MgADP Release and MgATP Binding Differently for the Two Cardiac Myosin Isoforms Expressed in Mouse Myocardium

Transgenic mouse models lacking myosin binding protein-C (cMyBP-C) have been used extensively to examine the influence of cMyBP-C on myocardial performance. Mouse ventricles normally express predominately α-myosin heavy chain (α-MyHC), while cMyBP-C knockout models (-/- and t/t) express a mixture of α- and β-MyHC isoforms. Performance comparisons between control mice and these knockouts are complicated by differences in MyHC isoform profile. We tested whether cMyBP-C differentially affected MgATP-dependent kinetics in the two cardiac MyHC isoforms. We applied varying MgATP concentrations to chemically-skinned myocardial strips undergoing length perturbation analysis and were able to deduce acto-myosin crossbridge MgADP release rate (k-ADP) and MgATP binding rate (k+ATP). Knockout mice treated for 10 days with L-thyroxine (t/tT4) expressed 90/10% α/β-MyHC as did transgenic controls expressing wildtype cMyBP-C (WTt/t-T4). Values for k-ADP were not different between t/tT4 (95.7±3.5 s−1) and WTt/t-T4 (90.3±9.6 s−1), but k+ATP was significantly lower in the t/tT4(154±4 mM−1s−1) compared to WTt/t-T4 (302±18 mM−1s−1), P<0.01. Mice fed a diet of 6-n-propyl-2-thiouracil (PTU) expressed 100% β-MyHC. Values for k-ADP were significantly higher in t/tPTU (24.8±1.0 s−1) compared to non-transgenic controls (NTGPTU, 15.6±1.3 s−1), P<0.01, but k+ATP was not significantly different in t/tPTU (77.5±14.5 mM−1s−1) compared to NTGPTU (99.8±20.0 mM−1s−1). These findings in the structural context of an intact cardiac sarcomere suggest that the presence of cMyBP-C influences MgATP-dependent kinetics differently for the two cardiac myosin isoforms. cMyBP-C does not influence MgADP release rate of α-MyHC, but effectively doubles the MgATP binding rate. In contrast, the presence of cMyBP-C reduces the MgADP release rate in the β-MyHC isoform, but does not change MgATP binding kinetics.

A Metal Switch for Controlling the Activity of Molecular Motor Proteins

Molecular motors, such as kinesins and myosins, as well as molecular switches such as G-proteins, use the energy from nucleotide hydrolysis to carry out cellular tasks. In addition to the P-loop, these proteins use similar structural motifs, called switch 1 and switch 2, to sense and respond to the presence or absence of the gamma-phosphate of the nucleotides and coordinate nucleotide hydrolysis. We have developed a strategy to probe metal interactions within kinesin and myosin molecular motors, by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine (-OH) and cysteine (−SH). We present the crystal structure of a kinesin motor domain bound to MnADP and report on a serine-to-cysteine substitution in the switch 1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn(II) to Mg(II). This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg(II). In kinesin-1, kinesin-5, kinesin-10 and kinesin-14, this defect is rescued by Mn(II), providing a way to control both the enzymatic activity and force-generating ability of these nanomachines. We also present ATPase data for an analogous substitution in the switch 1 of non-muscle myosin-2 from Dictyostelium discoideum. This mutant myosin shows aberrant actin interaction whereby actomyosin dissociation becomes slow and rate-limiting in the presence of Mg(II), yet is rescued to wild-type activity by Mn(II). These data are consistent with switch 1 being unable to efficiently close upon MgATP binding, thus affecting the opening of the upper 50K domain. There are several relevant and important applications to this metal switch technology that will allow further biophysical characterization of molecular motors and molecular switch proteins.

Phosphorylation of Serine 282 in Cardiac Myosin Binding Protein-C Increases Myosin MgADP Release and MgATP Binding Rates in Mouse Myocardium

Cardiac myosin binding protein-C (cMyBP-C) modulates contractility in part through phosphorylation of serines (S) found in the cardiac-specific motif between domains C1 and C2. S282 phosphorylation appears to allow the hierarchical phosphorylation of S302 and S307 by protein kinase-A (PKA). We investigated whether S282 phosphorylation status influenced the effects of PKA on acto-myosin cross-bridge MgADP release rate (k-ADP) and MgATP binding rate (k+ATP) in myocardial strips. Transgenic mice expressing cMyBP-C with a single amino acid switch at S282 to glutamic acid (D) or alanine (A) were generated to mimic phosphorylated or unphosphorylated states, respectively. Mice were fed L-thyroxin for 10 days to ensure similar (>90%) α-myosin heavy chain (α-MyHC) background. Skinned papillary muscles were pretreated with alkaline phosphatase to reduce any differential in vivo phosphorylation, and examined by length perturbation analysis at maximum calcium activation (pCa 4.8) as MgATP concentration varied (0 to 5 mM). α-MyHC k-ADP and k+ADP increased 90% (212±22 vs. 111±9 s−1) and 45% (867±93 vs. 595±50 mM−1 s−1) for S282D vs. S282A, demonstrating that cMyBP-C S282 phosphorylation status significantly affects α-MyHC kinetics. Following PKA treatment, k-ADP and k+ADP values did not differ between S282D and S282A, due to PKA reducing k-ADP by 23% (163±19 s−1) and k+ATP by 45% (475±35 mM−1 s−1) for S282D, but increasing k-ADP by 23% (137±9 s−1) and decreasing k+ATP by 23% (457±52 mM−1 s−1) for S282A. These results suggest that S282 phosphorylation most significantly affects the initial functional status of the myocardium, prior to PKA-mediated effects from cMyBP-C or other phosphorylatable myofilament proteins.

Importance of Aspartate Residues in Balancing the Flexibility and Fine-Tuning the Catalysis of Human 3-Phosphoglycerate Kinase

The exact role of the metal ion, usually Mg(2+), in the catalysis of human 3-phosphoglycerate kinase, a well-studied two-domain enzyme, has not been clarified. Here we have prepared single and double alanine mutants of the potential metal-binding residues, D374 and D218. While all mutations weaken the catalytic interactions with Mg(2+), they surprisingly strengthen binding of both MgADP and MgATP, and the effects are even more pronounced for ADP and ATP. Thermodynamic parameters of binding indicate an increase in the binding entropy as a reason for the strengthening. In agreement with the experimental results, computer-simulated annealing calculations for the complexes of these mutants have supported the mobility of the nucleotide phosphates and, as a consequence, formation of their new interaction(s) within the active site. A similar type of mobility is suggested to be a characteristic feature of the nucleotide site of the wild-type enzyme, too, both in its inactive open conformation and in the active closed conformation. This mobility of the nucleotide phosphates that is regulated by the aspartate side chains of D218 and D374 through the complexing Mg(2+) is suggested to be essential in enzyme function.

NMR and Fluorescence Studies of Drug Binding to the First Nucleotide Binding Domain of SUR2A

ATP sensitive potassium (K(ATP)) channels are composed of four copies of a pore-forming inward rectifying potassium channel (Kir6.1 or Kir6.2) and four copies of a sulfonylurea receptor (SUR1, SUR2A, or SUR2B) that surround the pore. SUR proteins are members of the ATP-binding cassette (ABC) superfamily of proteins. Binding of MgATP at the SUR nucleotide binding domains (NBDs) results in NBD dimerization, and hydrolysis of MgATP at the NBDs leads to channel opening. The SUR proteins also mediate interactions with K(ATP) channel openers (KCOs) that activate the channel, with KCO binding and/or activation involving residues in the transmembrane helices and cytoplasmic loops of the SUR proteins. Because the cytoplasmic loops make extensive interactions with the NBDs, we hypothesized that the NBDs may also be involved in KCO binding. Here, we report nuclear magnetic resonance (NMR) spectroscopy studies that demonstrate a specific interaction of the KCO pinacidil with the first nucleotide binding domain (NBD1) from SUR2A, the regulatory SUR protein in cardiac K(ATP) channels. Intrinsic tryptophan fluorescence titrations also demonstrate binding of pinacidil to SUR2A NBD1, and fluorescent nucleotide binding studies show that pinacidil binding increases the affinity of SUR2A NBD1 for ATP. In contrast, the KCO diazoxide does not interact with SUR2A NBD1 under the same conditions. NMR relaxation experiments and size exclusion chromatography indicate that SUR2A NBD1 is monomeric under the conditions used in drug binding studies. These studies identify additional binding sites for commonly used KCOs and provide a foundation for testing binding of drugs to the SUR NBDs.