Formation and characterization of kinesin.ADP.fluorometal complexes

Recent crystallographic studies of motor proteins showed that the structure of the motor domains of myosin and kinesin are highly conserved. Thus, these motor proteins, which are important for motility, may share a common mechanism for generating energy from ATP hydrolysis. We have previously demonstrated that, in the presence of ADP, myosin forms stable ternary complexes with new phosphate analogues of aluminum fluoride (AlF(4)(-)) and beryllium fluoride (BeF(n)), and these stable complexes mimic the transient state along the ATPase kinetic pathway [Maruta et al. (1993) J. Biol. Chem. 268, 7093-7100]. In this study, we examined the formation of kinesin.ADP.fluorometals ternary complexes and analyzed their characteristics using the fluorescent ATP analogue NBD-ATP (2'(3')-O-[6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl]-ADP). Our results suggest that these ternary complexes may mimic transient state intermediates in the kinesin ATPase cycle. Thus, the kinesin.ADP.AlF(4)(-) complex resembles the kinesin.ADP state, and the kinesin.ADP.BeF(n) complex mimics the kinesin.ADP.P(i) state.     

Accurate determination of the oxidative phosphorylation affinity for ADP in isolated mitochondria

Background

Mitochondrial dysfunctions appear strongly implicated in a wide range of pathologies. Therefore, there is a growing need in the determination of the normal and pathological integrated response of oxidative phosphorylation to cellular ATP demand. The present study intends to address this issue by providing a method to investigate mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria.

Methodology/Principal Findings

The proposed method is based on the simultaneous monitoring of substrate oxidation (determined polarographically) and phosphorylation (determined using the glucose - hexokinase - glucose-6-phosphate dehydrogenase - NADP⁺ enzymatic system) rates, coupled to the determination of actual ADP and ATP concentrations by bioluminescent assay. This enzymatic system allows the study of oxidative phosphorylation during true steady states in a wide range of ADP concentrations. We demonstrate how the application of this method allows an accurate determination of mitochondrial affinity for ADP from both oxidation (K_mVox) and phosphorylation (K_mVp) rates. We also demonstrate that determination of K_mVox leads to an important overestimation of the mitochondrial affinity for ADP, indicating that mitochondrial affinity for ADP should be determined using phosphorylation rate. Finally, we show how this method allows the direct and precise determination of the mitochondrial coupling efficiency. Data obtained from rat skeletal muscle and liver mitochondria illustrate the discriminating capabilities of this method.

Conclusions/Significance

Because the proposed method allows the accurate determination of mitochondrial oxidative phosphorylation affinity for ADP in isolated mitochondria, it also opens the route to a better understanding of functional consequences of mitochondrial adaptations/dysfunctions arising in various physiological/pathophysiological conditions.     

Biochemical and structural studies on the high affinity of Hsp70 for ADP

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the β-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.     

ADP regulates movements of mitochondria in neurons

Mitochondria often reside in subcellular regions with high metabolic demands. We examined the mechanisms that can govern the relocation of mitochondria to these sites in respiratory neurons. Mitochondria were visualized using tetramethylrhodamineethylester, and their movements were analyzed by applying single-particle tracking. Intracellular ATP ([ATP](i)) was assessed by imaging the luminescence of luciferase, the fluorescence of the ATP analog TNP-ATP, and by monitoring the activity of K(ATP) channels. Directed movements of mitochondria were accompanied by transient increases in TNP-ATP fluorescence. Application of glutamate and hypoxia reversibly decreased [ATP](i) levels and inhibited the directed transport. Injections of ATP did not rescue the motility of mitochondria after its inhibition by hypoxia. Introduction of ADP suppressed mitochondrial movements and occluded the effects of subsequent hypoxia. Mitochondria decreased their velocity in the proximity of synapses that correlated with local [ATP](i) depletions. Using a model of motor-assisted transport and Monte Carlo simulations, we showed that mitochondrial traffic is more sensitive to increases in [ADP](i) than to [ATP](i) depletions. We propose that consumption of synaptic ATP can produce local increases in [ADP](i) and facilitate the targeting of mitochondria to synapses.     

Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity

Kinesin binds to microtubules with half-site ADP release to form a tethered intermediate with one attached head without nucleotide and one tethered head that retains its bound ADP. For DKH405 containing amino acid residues 1-405 of Drosophila kinesin, release of the remaining ADP from the tethered head is slow (0.05 s(-1)), but release is accelerated by added ADP or ATP. The maximum rate of ADP-stimulated dissociation of tethered DKH405 from the microtubule is approximately 12 s(-1) as determined by turbidity. Parallel measurements of ADP-stimulated release of 2'(3')-O-(N-methylanthraniloyl)-ADP (mantADP) from the tethered intermediate by fluorescence indicate that the reaction is biphasic with a fast phase that occurs at a rate that is similar to dissociation. The rate of the slow phase is dependent on the concentrations of salt and microtubules and is equal in each case to the rate for bimolecular stimulation of ADP release by microtubules as measured independently. These results are consistent with a scheme in which the fast phase, with approximately one-third of the total amplitude change, is due to ADP-stimulated release of mantADP from the tethered intermediate at approximately 6 s(-1). This direct release of mantADP continues until terminated by dissociation of DKH405 from the microtubule at approximately 12 s(-1). The majority of the amplitude change thus occurs through bimolecular recombination of DKH405.mantADP with microtubules following initial dissociation. Analysis of a simple scheme indicates that hydrolysis of ATP at the attached head before the tethered head can release its ADP and become tightly bound may be the principal limitation to processivity.     

Modulation of kinesin half-site ADP release and kinetic processivity by a spacer between the head groups

A series of modifications of the junction of the neck linker and neck coil of dimeric Drosophila kinesin were constructed to determine the influence of head orientation and spacing on the ATPase kinetics. Ala(345) is the first residue in the coiled-coil of the neck, and its replacement with glycine or proline produces no significant change in the k(cat) or K(0.5(MT)) values for activation of their ATPase by microtubules (MTs) or in their k(bi(ratio)) value for the average number of ATP molecules hydrolyzed during a processive encounter with a MT. Addition or deletion of a single amino acid at the junction produces only modest changes with less than a 2-fold reduction in kinetic processivity. Insertion of a spacer of 6 or 12 additional amino acids at the neck linker junction increases the K(0.5(MT)) value by 3-4-fold with a corresponding decrease in kinetic processivity. The sliding velocities of all the mutant constructs under multimotor conditions are within 30% of the wild-type value. All the constructs with single residue changes exhibit half-site ADP release on binding to MTs. The constructs with long insertion, however, rapidly release both ADP molecules per dimer on binding to a MT, indicating that the steric constraints that prevent release of ADP from the tethered head of wild-type kinesin have been relieved by the long insertions. The constructs with long inserts have decreased kinetic processivity and dissociate from the MT during ATP hydrolysis 3-fold faster than wild-type.     

The phosphorylation of kinesin regulates its binding to synaptic vesicles.

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.     

When a motor goes bad: a kinesin regulates neuronal survival

In this issue of Cell, Midorikawa et al. (2006) demonstrate that the kinesin superfamily member KIF4, a microtubule-based molecular motor, regulates the survival of electrically active neurons in the developing brain by modulating the function of poly(ADP-ribose) polymerase-1 in an unexpected way.     

The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.     

HURP regulates chromosome congression by modulating kinesin Kif18A function

Chromosome biorientation and congression during mitosis require precise control of microtubule dynamics [1-4]. The?dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of microtubule-associated proteins (MAPs) [4-9]. Recently, a MAP known as HURP (hepatoma upregulated protein) was identified [10-12]. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP) releases HURP from the importin β inhibitory complex and allows it to localize to the kinetochore fiber (k-fiber) [12, 13]. HURP stabilizes k-fibers and promotes chromosome congression [12, 14, 15]. However, the molecular mechanism underlying the role of HURP in regulating chromosome congression remains elusive. Here, we show that overexpression of the N-terminal microtubule binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore, quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our results demonstrate in part the regulatory mechanism for Kif18A during chromosome congression and provide new insights into the mechanism of chromosome movement at the metaphase plate.     

A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes

The kinesin-13 motor, KLP10A, destabilizes microtubules at their minus ends in mitosis and binds to polymerizing plus ends in interphase, regulating spindle and microtubule dynamics. Little is known about kinesin-13 motors in meiosis. In this study, we report that KLP10A localizes to the unusual pole bodies of anastral Drosophila melanogaster oocyte meiosis I spindles as well as spindle fibers, centromeres, and cortical microtubules. We frequently observe the pole bodies attached to cortical microtubules, indicating that KLP10A could mediate spindle anchoring to the cortex via cortical microtubules. Oocytes treated with drugs that suppress microtubule dynamics exhibit spindles that are reoriented more vertically to the cortex than untreated controls. A dominant-negative klp10A mutant shows both reoriented and shorter oocyte spindles, implying that, unexpectedly, KLP10A may stabilize rather than destabilize microtubules, regulating spindle length and positioning the oocyte spindle. By altering microtubule dynamics, KLP10A could promote spindle reorientation upon oocyte activation.     

A conserved tyrosine in the neck of a fungal kinesin regulates the catalytic motor core

The neck domain of fungal conventional kinesins displays characteristic properties which are reflected in a specific sequence pattern. The exchange of the strictly conserved Tyr 362, not present in animals, into Lys, Cys or Phe leads to a failure to dimerize. The destabilizing effect is confirmed by a lower coiled-coil propensity of mutant peptides. Whereas the Phe substitution has only a structural effect, the Lys and Cys replacements lead to dramatic kinetic changes. The steady state ATPase is 4- to 7-fold accelerated, which may be due to a faster microtubule-stimulated ADP release rate. These data suggest that an inhibitory effect of the fungal neck domain on the motor core is mediated by direct interaction of the aromatic ring of Tyr 362 with the head, whereas the OH group is essential for dimerization. This is the first demonstration of a direct influence of the kinesin neck region in regulation of the catalytic activity.     

JIP1 regulates the directionality of APP axonal transport by coordinating kinesin and dynein motors

Regulation of the opposing kinesin and dynein motors that drive axonal transport is essential to maintain neuronal homeostasis. Here, we examine coordination of motor activity by the scaffolding protein JNK-interacting protein 1 (JIP1), which we find is required for long-range anterograde and retrograde amyloid precursor protein (APP) motility in axons. We identify novel interactions between JIP1 and kinesin heavy chain (KHC) that relieve KHC autoinhibition, activating motor function in single molecule assays. The direct binding of the dynactin subunit p150^Glued to JIP1 competitively inhibits KHC activation in vitro and disrupts the transport of APP in neurons. Together, these experiments support a model whereby JIP1 coordinates APP transport by switching between anterograde and retrograde motile complexes. We find that mutations in the JNK-dependent phosphorylation site S421 in JIP1 alter both KHC activation in vitro and the directionality of APP transport in neurons. Thus phosphorylation of S421 of JIP1 serves as a molecular switch to regulate the direction of APP transport in neurons.     

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase

The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.     

An inhibitory high affinity binding site for ADP in the oligomycin-sensitive ATPase of beef heart submitochondrial particles.

Kinetic evidence are presented for the existence of a high affinity inhibitory site for ADP /K_i < 10^?7 M/ in the oligomycin-sensitive ATPase of beef heart submitochondrial particles. The ATPase·ADP complex is completely inactive in the ATPase reaction; it can be converted into active ATPase in a slow ATP-dependent reaction. The dependence of a first order rate constant for activation of the enzyme·ADP complex on concentration of ATP gives a K_m value equal to that for ATP in the ATPase reaction. The data obtained suggest that the membrane-bound ATPase complex contains two kinetically distinct nucleotide-binding centers, i.e. center 1 binds ATP or ADP with a formation of enzyme-substrate or enzyme-competitive inhibitor complexes: center 2 binds ADP with a formation of a complex which is able to bind ATP in center 1 and unable to hydrolyze the bound ATP. The binding of ATP or ADP in center 1 changes the reactivity of center 2 towards ADP.     

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.     

Archaeal histone tetramerization determines DNA affinity and the direction of DNA supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.