The creatine kinase reaction: a simple reaction with functional complexity

The classical role of PCr is seen as a reservoir of high-energy phosphates defending cellular ATP levels under anaerobic conditions, high rates of energy transfer or rapid fluctuations in energy requirement. Although the high concentration of PCr in glycolytic fast-twitch fibers supports the role of PCr as a buffer of ATP, the primary importance of the creatine kinase (CK) reaction may in fact be to counteract large increases in ADP, which could otherwise inhibit cellular ATPase-mediated systems. A primary role for CK in the maintenance of ADP homeostasis may explain why, in many conditions, there is an inverse relationship between PCr and muscle contractility but not between ATP and muscle contractility. The high rate of ATP hydrolysis during muscle contraction combined with restricted diffusion of ADP suggests that ADP concentration increases transiently during the contraction phase (ADP spikes) and that these are synchronized with the contraction. The presence of CK, structurally bound in close vicinity to the sites of ATP utilization, will reduce the amplitude and duration of the ADP spikes through PCr-mediated phosphotransfer. When PCr is reduced, the efficiency of CK as an ATP buffer will be reduced and the changes in ADP will become more prominent. The presence of ADP spikes is supported by the finding that other processes known to be activated by ADP (i.e. AMP deamination and glycolysis) are stimulated during exercise but not during anoxia, despite the same low global energy state. Breakdown of PCr is driven by increases in ADP above that depicted by the CK equilibrium and the current method to calculate ADPfree from the CK reaction in a contracting muscle is therefore questionable.     

Synphilin-1 is developmentally localized to synaptic terminals, and its association with synaptic vesicles is modulated by alpha-synuclein

Alpha-synuclein is the major component of Lewy bodies in patients with Parkinson's disease, and mutations in the alpha-synuclein gene are responsible for some familial forms of the disease. alpha-Synuclein is enriched in the presynapse, but its synaptic targets are unknown. Synphilin-1 associates in vivo with alpha-synuclein promoting the formation of intracellular inclusions. Additionally synphilin-1 has been found to be an intrinsic component of Lewy bodies in patients with Parkinson's disease. To understand the role of synphilin-1 in Parkinson's disease, we sought to define its localization and function in the brain. We now report that, like alpha-synuclein, synphilin-1 was enriched in neurons. In young rats, synphilin-1 was prominent in neuronal cell bodies but gradually migrated to neuropil during development. Immunoelectron microscopy of adult rat cerebral cortex demonstrated that synphilin-1 was highly enriched in presynaptic nerve terminals. Synphilin-1 co-immunoprecipitated with synaptic vesicles, indicating a strong association with these structures. In vitro binding experiments demonstrated that the N terminus of synphilin-1 robustly associated with synaptic vesicles and that this association was resistant to high salt washing but was abolished by inclusion of alpha-synuclein in the incubation medium. Our data indicated that synphilin-1 is a synaptic partner of alpha-synuclein, and it may mediate synaptic roles attributed to alpha-synuclein.     

Siah-1 facilitates ubiquitination and degradation of synphilin-1

Parkinson's disease is a common neurodegenerative disorder characterized by loss of dopaminergic neurons and appearance of Lewy bodies, cytoplasmic inclusions that are highly enriched with ubiquitin. Synphilin-1, alpha-synuclein, and Parkin represent the major components of Lewy bodies and are involved in the pathogenesis of Parkinson's disease. Synphilin-1 is an alpha-synuclein-binding protein that is ubiquitinated by Parkin. Recently, a mutation in the synphilin-1 gene has been reported in patients with sporadic Parkinson's disease. Although synphilin-1 localizes close to synaptic vesicles, its function remains unknown. To investigate the proteins that interact with synphilin-1, the present study performed a yeast two-hybrid screening and identified a novel interacting protein, Siah-1 ubiquitin ligase. Synphilin-1 and Siah-1 proteins were endogenously expressed in the central nervous system and were found to coimmunoprecipitate each other in rat brain homogenate. Confocal microscopic analysis revealed colocalization of both proteins in cells. Siah-1 was found to interact with the N terminus of synphilin-1 through its substrate-binding domain and to specifically ubiquitinate synphilin-1 via its RING finger domain. Siah-1 facilitated synphilin-1 degradation via the ubiquitin-proteasome pathway more efficiently than Parkin. Siah-1 was found to not facilitate ubiquitination and degradation of wild type or mutant alpha-synuclein. Synphilin-1 inhibited high K+-induced dopamine release from PC12 cells. Siah-1 was found to abrogate the inhibitory effects of synphilin-1 on dopamine release. Such findings suggest that Siah-1 might play a role in regulation of synphilin-1 function.     

Synphilin Isoforms and the Search for a Cellular Model of Lewy Body Formation in Parkinson's Disease

A common finding in many neurodegenerative diseases is the presence of inclusion bodies made of aggregated proteins in neurons of affected brain regions. In Parkinson's disease, the inclusion bodies are referred to as Lewy bodies and their main component is α-synuclein. Although many studies have suggested that inclusion bodies may be cell protective, it is still not clear whether Lewy bodies promote or inhibit dopaminergic cell death in Parkinson's disease. Synphilin-1 interacts with α-synuclein and is present in Lewy bodies. Accumulation of ubiquitylated synphilin-1 leads to massive formation of inclusion bodies, which resemble Lewy bodies by their ability to recruit α-synuclein. We have recently isolated an isoform of synphilin-1, synphilin-1A, that spontaneously aggregates in cells, and is present in detergent-insoluble fractions of brain protein samples from α-synucleinopathy patients. Synphilin-1A displays marked neuronal toxicity and, upon proteasome inhibition, accumulates into ubiquitylated inclusions with concomitant reduction of its intrinsic toxicity. The fact that α-synuclein interacts with synphilin-1A, and is recruited to synphilin-1A inclusion bodies in neurons together with synphilin-1, further indicates that synphilin-1A cell model is relevant for research on Parkinson's disease. Synphilin-1A cell model may help provide important insights regarding the role of inclusion bodies in Parkinson's disease and other neurodegenerative disorders.     

Aggregate formation and toxicity by wild-type and R621C synphilin-1 in the nigrostriatal system of mice using adenoviral vectors

Synphilin-1 was described as a protein interacting with α-synuclein and is commonly found in Lewy bodies, the pathological hallmark of Parkinson's disease (PD). Our group has previously described and characterized in vitro a mutation in the synphilin-1 gene (R621C) in PD patients. Providing the first characterization of synphilin-1 expression in an animal model, we here used adenoviral gene transfer to study the effects of wild-type (WT) and R621C synphilin-1 in dopaminergic neurons in mouse brain. As synphilin-1 is commonly used to trigger aggregation of α-synuclein in cell culture, we investigated not only non-transgenic C57Bl/6 mice but also A30P-α-synuclein transgenic animals. Both WT synphilin-1 and R621C synphilin-1 led to the formation of Thioflavine-S positive inclusions in C57Bl/6 mice and degeneration of dopaminergic neurons in the substantia nigra. R621C synphilin-1 induced more aggregate formation than WT synphilin-1 in A30P-α-synuclein transgenic mice, consistent with the role of the R621C mutation as a susceptibility factor for PD. Synphilin-1 expression may be used to improve current mouse models of PD, as it induced both the formation of aggregates and degeneration of dopaminergic neurons, two core characteristics of PD that have not been well reproduced with expression of α-synuclein.     

Casein kinase II-mediated phosphorylation regulates alpha-synuclein/synphilin-1 interaction and inclusion body formation

Alpha-synuclein is a phosphoprotein that accumulates as a major component of Lewy bodies in the brains of patients with Parkinson disease. Synphilin-1, which is also present in Lewy bodies, binds with alpha-synuclein and forms cytoplasmic inclusions in transfected cells. Yet the molecular determinants of this protein-protein interaction are unknown. Here we report that casein kinase II (CKII) phosphorylates synphilin-1 and that the beta subunit of this enzyme complex binds to synphilin-1. Additionally, both CKII alpha and beta subunits are present within cytoplasmic inclusions in cells that overexpress synphilin-1. Notably, the interaction between synphilin-1 and alpha-synuclein is markedly dependent on phosphorylation. Inhibition of CKII activity by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole blocks the binding between these two proteins and significantly reduces the percentage of cells that contain eosinophilic cytoplasmic inclusions. Mutation of the major CKII phosphorylation site in alpha-synuclein (S129A) has no significant impact on the binding between alpha-synuclein and synphilin-1 or on the formation of synphilin-1/alpha-synuclein-positive inclusions. These data suggest that the CKII-mediated phosphorylation of synphilin-1 rather than that of alpha-synuclein is critical in modulating their tendency to aggregate into inclusions. These observations collectively indicate that a ubiquitous post-translational modification such as phosphorylation can regulate inclusion body formation in the context of alpha-synuclein and synphilin-1 interaction.     

Dorfin localizes to Lewy bodies and ubiquitylates synphilin-1

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of nigra dopaminergic neurons. Lewy bodies (LBs) are a characteristic neuronal inclusion in PD brains. In this study, we report that Dorfin, a RING finger-type ubiquityl ligase for mutant superoxide dismutase-1, was localized with ubiquitin in LBs. Recently, synphilin-1 was identified to associate with alpha-synuclein and to be a major component of LBs. We found that overexpression of synphilin-1 in cultured cells led to the formation of large juxtanuclear inclusions, but showed no cytotoxicity. Dorfin colocalized in these large inclusions with ubiquitin and proteasomal components. In contrast to full-length synphilin-1, overexpression of the central portion of synphilin-1, including ankyrin-like repeats, a coiled-coil domain, and an ATP/GTP-binding domain, predominantly led to the formation of small punctate aggregates scattered throughout the cytoplasm and showed cytotoxic effects. Dorfin and ubiquitin did not localize in these small aggregates. Overexpression of the N or C terminus of synphilin-1 did not lead to the formation of any aggregates. Dorfin physically bound and ubiquitylated synphilin-1 through its central portion, but did not ubiquitylate wild-type or mutant alpha-synuclein. These results suggest that the central domain of synphilin-1 has an important role in the formation of aggregates and cytotoxicity and that Dorfin may be involved in the pathogenic process of PD and LB formation by ubiquitylation of synphilin-1.     

Probing the structural and energetic basis of kinesin-microtubule binding using computational alanine-scanning mutagenesis

Kinesin-microtubule (MT) binding plays a critical role in facilitating and regulating the motor function of kinesins. To obtain a detailed structural and energetic picture of kinesin-MT binding, we performed large-scale computational alanine-scanning mutagenesis based on long-time molecular dynamics (MD) simulations of the kinesin-MT complex in both ADP and ATP states. First, we built three all-atom kinesin-MT models: human conventional kinesin bound to ADP and mouse KIF1A bound to ADP and ATP. Then, we performed 30 ns MD simulations followed by kinesin-MT binding free energy calculations for both the wild type and mutants obtained after substitution of each charged residue of kinesin with alanine. We found that the kinesin-MT binding free energy is dominated by van der Waals interactions and further enhanced by electrostatic interactions. The calculated mutational changes in kinesin-MT binding free energy are in excellent agreement with results of an experimental alanine-scanning study with a root-mean-square error of ~0.32 kcal/mol [Woehlke, G., et al. (1997) Cell 90, 207-216]. We identified a set of important charged residues involved in the tuning of kinesin-MT binding, which are clustered on several secondary structural elements of kinesin (including well-studied loops L7, L8, L11, and L12, helices α4, α5, and α6, and less-explored loop L2). In particular, we found several key residues that make different contributions to kinesin-MT binding in ADP and ATP states. The mutations of these residues are predicted to fine-tune the motility of kinesin by modulating the conformational transition between the ADP state and the ATP state of kinesin.     

The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.     

All-atom molecular dynamics simulations reveal how kinesin transits from one-head-bound to two-heads-bound state

Kinesin dimer walks processively along a microtubule (MT) protofilament in a hand-over-hand manner, transiting alternately between one-head-bound (1HB) and two-heads-bound (2HB) states. In 1HB state, one head bound by adenosine diphosphate (ADP) is detached from MT and the other head is bound to MT. Here, using all-atom molecular dynamics simulations we determined the position and orientation of the detached ADP-head relative to the MT-bound head in 1HB state. We showed that in 1HB state when the MT-bound head is in ADP or nucleotide-free state, with its neck linker being undocked, the detached ADP-head and the MT-bound head have the high binding energy, and after adenosine triphosphate (ATP) binds to the MT-bound head, with its neck linker being docked, the binding energy between the two heads is reduced greatly. These results reveal how the kinesin dimer retains 1HB state before ATP binding and how the dimer transits from 1HB to 2HB state after ATP binding. Key residues involved in the head-head interaction in 1HB state were identified.     

Assessment of the rate of bound substrate interconversion and of ATP acceleration of product release during catalysis by mitochondrial adenosine triphosphatase

The oxygen exchange parameters for the hydrolysis of ATP by the F1-ATPase have been determined over a 140,000-fold range of ATP concentrations and a 5,000-fold range of reaction velocity. The average number of water oxygens incorporated into each Pi product ranges from a limit of about 1.02 at saturating ATP concentrations to a limit of about 3.97 at very low ATP concentrations. The latter value represents 400 reversals of hydrolysis of bound ATP prior to Pi dissociation. In accord with the binding change mechanism, this means that ATP binding at one catalytic site increases the off constant of Pi and ADP from another catalytic site by at least 20,000-fold, equivalent to the use of 6 kcal mol-1 of ATP binding energy to promote product release. The estimated rate of reversal of hydrolysis of F1-ATPase-bound ATP to bound ADP + Pi varies only about 5-fold with ATP concentration. The rate is similar that observed previously for reversal of bound ATP hydrolysis or synthesis with the membrane-bound enzyme and is greater than the rate of net ATP formation during oxidative phosphorylation. This adds to evidence that energy input or membrane components are not required for bound ATP synthesis.     

Synphilin-1 enhances α-synuclein aggregation in yeast and contributes to cellular stress and cell death in a Sir2-dependent manner

Background

Parkinson''s disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive.

Methodology/Principal Findings

We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation.

Conclusions/Significance

Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.     

Properties of ATP tightly bound to catalytic sites of chloroplast ATP synthase

Under steady state photophosphorylating conditions, each ATP synthase complex from spinach thylakoids contains, at a catalytic site, about one tightly bound ATP molecule that is rapidly labeled from medium 32Pi. The level of this bound [32P]ATP is markedly reduced upon de-energization of the spinach thylakoids. The reduction is biphasic, a rapid phase in which the [32P] ATP/synthase complex drops about 2-fold within 10 s, followed by a slow phase, kobs = 0.01/min. A decrease in the concentration of medium 32Pi to well below its apparent Km for photophosphorylation is required to decrease the amount of tightly bound ATP/synthase found just after de-energization and before the rapid phase of bound ATP disappearance. The [32P]ATP that remains bound after the rapid phase appears to be mostly at a catalytic site as demonstrated by a continued exchange of the oxygens of the bound ATP with water oxygens. This bound [32P]ATP does not exchange with medium Pi and is not removed by the presence of unlabeled ATP. The levels of tightly bound ADP and ATP arising from medium ADP were measured by a novel method based on use of [beta-32P]ADP. After photophosphorylation and within minutes after the rapid phase of bound ATP loss, the measured ratio of bound ADP to ATP was about 1.4 and the sum of bound ADP plus ATP was about 1/synthase. This ratio is smaller than that found about 1 h after de-energization. Hence, while ATP bound at catalytic sites disappears, bound ADP appears. The results suggest that during and after de-energization the bound ATP disappears from the catalytic site by hydrolysis to bound ADP and Pi with subsequent preferential release of Pi. These and related observations can be accommodated by the binding change mechanism for ATP synthase with participation of alternating catalytic sites and are consistent with a deactivated state arising from occupancy of one catalytic site on the synthase complex by an inhibitory ADP without presence of Pi.     

Uni-site ATP synthesis in thylakoids

Uni-site ATP synthesis was measured with thylakoids. The membrane-bound ATP-synthase, CF0F1, was brought into the active, reduced state by illumination in the presence of thioredoxin, dithiothreitol and phosphate. This enzyme contains two tightly bound ATP per CF0F1. ATP was released from the enzyme when ADP was added in substoichiometric amounts during illumination. Experiments with [14C]ADP indicated that after binding the same nucleotide was phosphorylated and released as [14C]ATP, i.e. only one site is involved in ATP-synthesis ('uni-site ATP-synthesis'). The two tightly bound ATP are not involved in the catalytic turnover. The rate constant for ADP binding was (4 +/- 2) x 10(6) M-1s-1. Compared to deenergized conditions the rate constant for ADP binding and that for ATP-release were drastically increased, i.e. membrane energization increased the rate constants for the ATP-synthesis direction.     

Protein substrates activate the ATP-dependent protease La by promoting nucleotide binding and release of bound ADP

The interaction of protein substrates with protease La from Escherichia coli enhances its ability to hydrolyze ATP and peptide bonds. These studies were undertaken to clarify how unfolded proteins allosterically stimulate this ATPase activity. The tetrameric protease can bind four molecules of ATP, which activates proteolysis, or four molecules of ADP, which inhibits enzymatic activity. Protein substrates stimulate binding of the nonhydrolyzable ATP analog [3H] adenyl-5'yl imidodiphosphate, although they do not increase the net binding of [3H]ATP or [3H]ADP. Once bound, ATP is quickly hydrolyzed to ADP, which remains noncovalently associated with protease La even through repeated gel filtrations. Exposure to protein substrates (e.g. denatured bovine serum albumin at 37 degrees C) induces the release of all the bound ADP from the enzyme. Nonhydrolyzable ATP analogs bound to the enzyme were not released by these substrates. Proteins that are not degraded (e.g. native bovine serum albumin) and oligopeptides that only bind to the catalytic site do not induce ADP release. Thus, polypeptide substrates have to interact with an allosteric site to induce this effect. The protein-induced ADP release is inhibited by high concentrations of Mg2+ and is highly temperature-dependent. Protein substrates promoted [3H]ATP binding in the presence of ADP and Mg2+ (i.e. ATP-ADP exchange) and reduced the ability of ADP to inhibit the enzyme's peptidase and ATPase activities. These results indicate that: 1) ADP release is a rate-limiting step in protease La function; 2) bound ADP molecules inhibit protein and ATP hydrolysis in vivo; 3) denatured proteins interact with the enzyme's regulatory site and promote ADP release, ATP binding, and their own hydrolysis.     

Removal of "tightly bound" nucleotides from soluble mitochondrial adenosine triphosphatase (F1).

Soluble mitochondrial ATPase (F1) from beef heart prepared in this laboratory contained approximately 1.8 mol of ADP and 0 mol of ATP/mol of F1 which were not removed by repeated precipitation of the enzyme with ammonium sulfate solution or by gel filtration in low ionic strength buffer containing EDTA. This enzyme had full coupling activity. Treatment of the enzyme with trypsin (5 mug/mg of F1 for 3 min) reduced the "tightly bound" ADP to zero, abolished coupling activity, but had no effect on the ATPase activity, stability, or membrane-binding capability of the F1. When the trypsin concentration was varied between 0 and 5 mug/mg of F1, tightly bound ADP was removed to varying degrees, and a correlation was seen between amount of residual tightly bound ADP and residual coupling activity. Gel filtration of the native F1 in high ionic strength buffer containing EDTA also caused complete loss of tightly bound ADP and coupling ability, whereas ATPase activity, stability, and membrane-binding capability were retained. The ADP-depleted F1 preparations were unable to rebind normal amounts of ADP or any ATP in simple reloading experiments. The results strongly suggest that tightly bound ADP is required for ATP synthesis and for energy-coupled ATP hydrolysis on F1. The results also suggest that ATP synthesis and energy-linked ATP hydrolysis rather than involving one nucleotide binding site on F1, involve a series or "cluster" of sites. The ATP hydrolysis site may represent one component of this cluster. The results show that nonenergy-coupled ATP hydrolysis on F1 can occur in the absence of tightly bound ADP or ATP.     

Mechanism of nucleotide binding to actomyosin VI: evidence for allosteric head-head communication

We have examined the kinetics of nucleotide binding to actomyosin VI by monitoring the fluorescence of pyrene-labeled actin filaments. ATP binds single-headed myosin VI following a two-step reaction mechanism with formation of a low affinity collision complex (1/K(1)' = 5.6 mm) followed by isomerization (k(+2)' = 176 s-1) to a state with weak actin affinity. The rates and affinity for ADP binding were measured by kinetic competition with ATP. This approach allows a broader range of ADP concentrations to be examined than with fluorescent nucleotide analogs, permitting the identification and characterization of transiently populated intermediates in the pathway. ADP binding to actomyosin VI, as with ATP binding, occurs via a two-step mechanism. The association rate constant for ADP binding is approximately five times greater than for ATP binding because of a higher affinity in the collision complex (1/K(5b)' = 2.2 mm) and faster isomerization rate constant (k(+5a)' = 366 s(-1)). By equilibrium titration, both heads of a myosin VI dimer bind actin strongly in rigor and with bound ADP. In the presence of ATP, conditions that favor processive stepping, myosin VI does not dwell with both heads strongly bound to actin, indicating that the second head inhibits strong binding of the lead head to actin. With both heads bound strongly, ATP binding is accelerated 2.5-fold, and ADP binding is accelerated >10-fold without affecting the rate of ADP release. We conclude that the heads of myosin VI communicate allosterically and accelerate nucleotide binding, but not dissociation, when both are bound strongly to actin.     

Critical role of specific ions for ligand-induced changes regulating pyruvate dehydrogenase kinase isoform 2

In the complete absence of K+ and phosphate (Pi), pyruvate dehydrogenase kinase isoform 2 (PDHK2) was catalytically very active but with an elevated Km for ATP, and this activity is insensitive to effector regulation. We find that K+ or 5-fold lower levels of NH4+ markedly enhanced quenching of Trp383 fluorescence of PDHK2 by ADP and ATP. K+ binding caused an approximately 40-fold decrease in the equilibrium dissociation constants (Kd) for ATP from approximately 120 to 3.0 microM and an approximately 25-fold decrease in Kd for ADP from approximately 950 to 38 microM. Linked reductions in Kd of PDHK2 for K+ were from approximately 30 to approximately 0.75 mM with ATP bound and from approximately 40 to approximately 1.7 mM with ADP bound. Without K+, there was little effect of ADP on pyruvate binding, but with 100 mM K+ and 100 microM ADP, the L0.5 of PDHK2 for pyruvate was reduced by approximately 14 fold. In the absence of K+, Pi had small effects on ligand binding. With 100 mM K+, 20 mM Pi modestly enhanced binding of ADP and hindered pyruvate binding but markedly enhanced the binding of pyruvate with ADP; the L0.5 for pyruvate was specifically decreased approximately 125-fold with 100 microM ADP. Pi effects were minimal when NH4+ replaced K+. We have quantified coupled binding of K+ with ATP and ADP and elucidated how linked K+ and Pi binding are required for the potent inhibition of PDHK2 by ADP and pyruvate.     

Structural insight into AMPK regulation: ADP comes into play

The AMP-activated protein kinase (AMPK), a sensor of cellular energy status found in all eukaryotes, responds to changes in intracellular adenosine nucleotide levels resulting from metabolic stresses. Here we describe crystal structures of a heterotrimeric regulatory core fragment from Schizosaccharomyces pombe AMPK in complex with ADP, ADP/AMP, ADP/ATP, and 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranotide (AICAR phosphate, or ZMP), a well-characterized AMPK activator. Prior crystallographic studies had revealed a single site in the gamma subunit that binds either ATP or AMP within Bateman domain B. Here we show that ZMP binds at this site, mimicking the binding of AMP. An analogous site in Bateman domain A selectively accommodates ADP, which binds in a distinct manner that also involves direct ligation to elements from the beta subunit. These observations suggest a possible role for ADP in regulating AMPK response to changes in cellular energy status.