mantATP Binding Research Articles

Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein.

South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD600 of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS–PAGE. The secondary structure of SACMV MP presented as predominantly β-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2′ (3′)-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.

The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy

Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.

The Regulation of ATP-Binding, PI Release and ADP Dissociation from Myosin II in Native Cardiac Myofibrils by the N-Terminal Extension of Cardiac Troponin T

Cardiac troponin T (cTnT) has an amino-terminal variable region that plays a role in modifying the overall protein conformation and functions in the calcium-dependent regulation of cardiac muscle contraction. Previous studies have shown that the N-terminal domain of cTnT mainly affects the tropomyosin binding site 1 that binds to the head-tail junction of tropomyosins in thin filament, a moiety involved in the cooperativity of the myofilaments upon calcium activation. It has also been shown that in comparison with wild type controls, transgenic mouse hearts over-expressing cTnT lacking the N-terminal variable region (cTnT-ND) had a decreased rate of contraction, which specifically elongates the rapid ejection phase while keeping total ejection time unchanged, suggesting that the altered cTnT molecule affects the actomyosin crossbridge kinetics. In the present study, we employed stopped flow analysis of mant-ATP binding, ADP dissociation and phosphate release in order to investigate which step(s) of the ATPase cycle is regulated by the N-terminal variable region of cTnT. The results showed that cTnT-ND myofibrils exhibited a maximum mant-ATP binding rate constant (k2) significantly lower than that of WT control, while the equilibrium constant K1 for the initial actomyosin complex formation remained unchanged over calcium concentration. ADP dissociation and Pi release from cTnT-ND myofibrils showed no significant change with increasing calcium concentration, however ADP release is slower in cTnT-ND myofibrils when compared to WT control. These results provide novel information regarding the function of the N-terminal variable region in modulating specific kinetic steps and selectively altering systolic but not diastolic function of the heart.

The Regulation of Actomyosin ATPase in Cardiac Muscle by the N-Terminal Extension of Cardiac Troponin T

Cardiac troponin T (cTnT) and cardiac troponin I (cTnI) both have an amino-terminal variable region that plays a role in modifying the overall protein conformation and functions in the calcium-dependent regulation of cardiac muscle function. Previous studies have showed that in comparison with wild type controls, transgenic mouse hearts over-expressing cTnT lacking the N-terminal variable region (cTnT-ND) had an increased rate of relaxation. In contrast, transgenic mouse heart over-expressing cTnI lacking the N-terminal extension (cTnI-ND) had lower sensitivity to calcium activation of ATPase, resulting enhanced ventricular relaxation and Frank-Starling response. Recently, we demonstrated that the second order mant-dATP binding rate of cardiac myofibrils containing cTnI-ND was three-fold as fast as that of wild type myofibrils in low [Ca2+]. The ADP dissociation rate of cTnI-ND myofibrils was positively dependent on calcium concentrations, while the wild type controls were not significantly affected. These results from native cardiac myofibrils under physiological conditions indicate that the N-terminal extension of cTnI plays a role in the calcium regulation of the kinetics of actomyosin ATPase. In the present study, we employed the same techniques in order to investigate which step(s) of the ATPase cycle is regulated by the N-terminal variable region of cTnT. mant-ATP binding, mant-ADP/ADP dissociation, and phosphate releasing rate of cTnT-ND myofibrils are examined and the results will provide novel information dissecting the function of the two troponin subunits and their posttranslational modifications.

Kinetic Characterization of Rice Plant Specific Kinesin E11 using Fluorescent ATP Analogue

Kinesin is an ATP-driven motor protein that plays important physiological roles in intracellular transport, mitosis and meiosis, control of microtubule dynamics, and signal transduction. Kinesin species derived from vertebrates have been well characterized. In contrast, plant specific kinesin have yet to be adequately characterized. We have previously demonstrated that some kinesins derived from rice plant have unique biochemical characteristic properties and structures.In this study, we characterized rice plant specific kinesin E11 that belongs to the plant specific At1 subfamily in kinesin-7 family. E11 motor domain was expressed by E. coli expression system and purified with Co-chelate column in order to characterize biochemical and ATPase kinetic properties. The fluorescent ATP analogues, Mant-ATP was employed for the kinetic characterization. We have successfully observed significant FRET between Mant-ATP and intrinsic tryptophan (Trp23) residue in E11. The kinetic parameters of initial binding of Mant-ATP to E11 and release of Mant-ADP from E11 were analyzed by monitoring the FRET using stopped flow apparatus and compared with other rice kinesins and conventional kinesin. The results revealed that the initial binding of ATP to E11 and release of ADP are slower than those of other rice plant specific kinesin.

Strain-Dependent Regulation of the Kinesin-1's Catalytic Activity as Studied by Disulfide-Crosslinking of the Neck Linker

Kinesin-1 walks along microtubules by alternately hydrolyzing ATP and moving two motor domains. Several recent studies have suggested that the strain developed through the neck linker is essential for the coordination between two motor domains, although the mechanism for the regulation of the motor domain's activity remains unknown. At the last annual meeting we employed disulfide-crosslinking between cysteine residues introduced into the neck linker and the motor domain to constrain the neck linker of a monomer in the forward or backward extended conformation, and using single molecule fluorescent observation we showed that detachment rate from the microtubule dramatically decreased when the neck linker is constrained in a backward extended conformation. To further investigate the effect of crosslinking on the ATP hydrolysis kinetics, we measured mant-ADP release after rapid mixing with microtubule and subsequent mant-ATP binding rates of the monomers. Stopped flow measurements showed that ADP release and ATP binding rates did not change after constraining in the backward extended conformation, suggesting that either ATP hydrolysis or Pi release step may be the rate-limiting. When the neck linker is constrained in a forward extended conformation, ATP binding rate did not changed but the ADP release rate dramatically decreased. These results suggest that ATP hydrolysis cycle of the motor domain can be differently regulated depending on the direction of the neck linker tension, explaining the alternate catalysis in the dimer.

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Kinesin Kar3Cik1 ATPase Pathway for Microtubule Cross-linking

Kar3Cik1 is a Saccharomyces cerevisiae kinesin-14 that functions to shorten cytoplasmic microtubules (MTs) during yeast mating yet maintains mitotic spindle stability by cross-linking anti-parallel interpolar MTs. Kar3 contains both an ATP- and a MT-binding site, yet there is no evidence of a nucleotide-binding site in Cik1. Presteady-state and steady-state kinetic experiments were pursued to define the regulation of Kar3Cik1 interactions with the MT lattice expected during interpolar MT cross-linking. The results reveal that association of Kar3Cik1 with the MT occurs at 4.9 μM(-1) s(-1), followed by a 5-s(-1) structural transition that limits ADP release from the Kar3 head. Mant-ATP binding occurred at 2.1 μM(-1) s(-1), and the pulse-chase experiments revealed an ATP-promoted isomerization at 69 s(-1). ATP hydrolysis was observed as a rapid step at 26 s(-1) and was required for the Kar3Cik1 motor to detach from MT. The conformational change at 5 s(-1) that occurred after Kar3Cik1 MT association and prior to ADP release was hypothesized to be the rate-limiting step for steady-state ATP turnover. We propose a model in which Kar3Cik1 interacts with the MT lattice through an alternating cycle of Cik1 MT collision followed by Kar3 MT binding with head-head communication between Kar3 and Cik1 modulated by the Kar3 nucleotide state and intramolecular strain.

Mechanistic Analysis of Kar3Cik1 for Mitotic Function

Kar3Cik1 is a S. cerevisae Kinesin-14 motor protein that functions to shorten cytoplasmic microtubule (MT) during yeast mating for nuclear fusion, yet crosslinks interpolar MTs (ipMTs) during anaphase. The Kar3 head contains both an ATP and MT binding site, yet the Cik1 head lacks an ATP catalytic site. Presteady-state and steady state experiments were conducted to define the mechanochemical pathway by which Kar3Cik1 stabilizes anti-parallel ipMTs for its mitotic function. To initiate the cycle, we used a high ADP strategy to promote MT-binding by the Cik1 head at 4.9 ∈1/4M-1s-1. The initial association is then followed by a 4-5 s-1 conformational change to induce Kar3 head binding to the MT with rapid ADP release from the active site at 109 s-1. MantATP binding to the nucleotide free MT•Kar3Cik1 is fast at 2.1 ∈1/4M-1s-1 with koff = 16.6 s-1. Pulse-chase methodology further reveals that MgATP binding to MT•Kar3Cik1 follows a two step process, formation of a collision complex followed by a 64 s-1 isomerization step. ATP hydrolysis occurs at 26 s-1 followed by motor detachment from microtubule at 11.5 s-1. The rate-limiting step for steady-state ATP turnover at 5 s-1 is hypothesized to be the conformational change leading to Kar3 head binding to MT. These initial results suggest a model in which Kar3Cik1 interacts with the MT through an alternating cycle of Cik1 binding followed by Kar3 binding. Because Cik1 does not have a nucleotide binding site, we propose that head-head communication is mediated by a strain-dependent mechanism. Supported by NIH GM54141.

ADP but Not Pi Dissociation Contributes to Rate Limitation for Escherichia coli Rho

To define the molecular mechanism by which ATP hydrolysis powers the 5'-->3' travel of homohexameric Escherichia coli transcription termination factor Rho along RNA, rates for association and dissociation of non-RNA substrates and products were measured. Rapid mix/chemical quench and stopped-flow spectrofluorometry measurements were carried out with Rho and [gamma-(32)P]ATP, mantADP, or fluorescently tagged E. coli phosphate-binding protein. The results indicate that the P(i) off-rate is not rate limiting, but at approximately 90 s(-1), the ADP dissociation rate is comparable to the 30 s(-1) k(cat). Previous results indicate that the chemistry step of ATP hydrolysis by Rho is at least 10-fold faster than the overall catalytic cycle. The as yet unmeasured RNA dissociation step, which could be associated with a protein conformation change, might also be a rate-limiting factor.

How myosin VI coordinates its heads during processive movement

A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for myosin VI, since its lever arm positions are reversed. Here, we demonstrate that myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily--kinesin I.

Dimeric Eg5 Maintains Processivity through Alternating-site Catalysis with Rate-limiting ATP Hydrolysis

Eg5/KSP is a homotetrameric, Kinesin-5 family member whose ability to cross-link microtubules has associated it with mitotic spindle assembly and dynamics for chromosome segregation. Transient-state kinetic methodologies have been used to dissect the mechanochemical cycle of a dimeric motor, Eg5-513, to better understand the cooperative interactions that modulate processive stepping. Microtubule association, ADP release, and ATP binding are all fast steps in the pathway. However, the acid-quench analysis of the kinetics of ATP hydrolysis with substrate in excess of motor was unable to resolve a burst of product formation during the first turnover event. In addition, the kinetics of P(i) release and ATP-promoted microtubule-Eg5 dissociation were observed to be no faster than the rate of ATP hydrolysis. In combination the data suggest that dimeric Eg5 is the first kinesin motor identified to have a rate-limiting ATP hydrolysis step. Furthermore, several lines of evidence implicate alternating-site catalysis as the molecular mechanism underlying dimeric Eg5 processivity. Both mantATP binding and mantADP release transients are biphasic. Analysis of ATP hydrolysis through single turnover assays indicates a surprising substrate concentration dependence, where the observed rate is reduced by half when substrate concentration is sufficiently high to require both motor domains of the dimer to participate in the reaction.

Vertebrate Myosin VIIb Is a High Duty Ratio Motor Adapted for Generating and Maintaining Tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

Nucleotide Binding Induces Conformational Changes in Escherichia coli Transcription Termination Factor Rho

The Escherichia coli Rho protein uses the energy of ATP binding and hydrolysis to translocate along RNA and cause transcription termination. Using fluorescence stopped-flow kinetic studies, we have discerned the conformational changes in the Rho protein that occur upon nucleotide and nucleic acid binding. We show that the 2', (3')-O-[N-methylanthraniloyl] derivative of ATP (mant-ATP) is a good fluorescent substrate of Rho and is hydrolyzed with a K(m) comparable with that for ATP but a k(cat) five to six times slower than that for ATP. The kinetics of ATP and mant-ATP binding indicates that, in the absence of RNA, the Rho protein is structurally distinct from the Rho hexamer found when bound to RNA or DNA. In the absence of RNA, the nucleotide-binding rates are 50- to 70-fold slower, and the dissociation rates are 40- to 120-fold slower than the corresponding rates in the presence of RNA. We conclude that RNA or DNA binding to the primary nucleic acid binding sites causes conformational changes in the Rho hexamer that result in the opening of the subunit interfaces. Furthermore, the kinetic studies revealed a unique protein conformational change in the Rho.RNA complex upon ATP binding that is a result of RNA contacting the secondary nucleic acid binding sites in the central channel of the Rho ring. This conformational change seems to render the Rho ring competent in ATP hydrolysis and translocation.

Kinetics of the E. coli replication factor DnaC protein-nucleotide interactions. II. Fluorescence anisotropy and transient, dynamic quenching stopped-flow studies of the reaction intermediates.

The nature of the intermediates in the binding of MANT-ATP and MANT-ADP to the E. coli replicative factor DnaC protein (accompanying paper) has been examined using the fluorescence intensity, anisotropy, and transient dynamic quenching stopped-flow techniques. Using molar fluorescence intensities of individual intermediates of the reaction, we derived the Stern-Volmer equation that provides a direct method to quantitatively address the quenching of the fluorescence of a transient intermediate by an external, neutral quencher. The data indicate that in the first intermediate, (C)(1), the solvent has full access to the MANT group. Thus, the nucleotide-binding site is located on the surface of the protein, fully open to the solvent. Moreover, formation of the first intermediate does not affect the structure of the binding site. On the other hand, in the second intermediate, (C)(2), the entire binding site changes its conformation, resulting in diminished access of the solvent to the bound nucleotide. The time course of the fluorescence anisotropy in the reaction provides direct, unique insight into the mobility of bound nucleotides in each intermediate. The analysis is facilitated by the fact that the anisotropy can be expressed as a function of the relative molar intensities and steady-state anisotropies of the individual intermediates. The major decrease of the nucleotide mobility occurs in the formation of the first intermediate and reflects the fact that the MANT group is immobilized to a similar extent as the ribose region of the bound nucleotides. Transition to the second intermediate and closing of the binding site leads to only a moderate, additional decrease of nucleotide mobility. The temperature effect on the studied interactions indicates that the formation of individual intermediates is accompanied by very different enthalpy and entropy changes predominantly generated from the structural changes of the protein. Analysis of the salt effect indicates that the net release of a single ion, observed in equilibrium studies, occurs in the formation of the first intermediate. The lack of any salt effect on the (C)(1) <--> (C)(2) transition indicates that the closing of the binding site does not include a net ion release or uptake. Moreover, prior to the nucleotide binding, the conformational transition of the DnaC protein is exclusively controlled by the nucleotide binding and release.

Insights into nucleotide binding in protein kinase A using fluorescent adenosine derivatives.

The binding of the methylanthraniloyl derivatives of ATP (mant-ATP), ADP (mant-ADP), 2'deoxyATP (mant-2'deoxyATP), and 3'deoxyATP (mant-3'deoxyATP) to the catalytic subunit of protein kinase A was studied to gain insights into the mechanism of nucleotide binding. The binding of the mant nucleotides leads to a large increase in fluorescence energy transfer at 440 nm, allowing direct measurements of nucleotide affinity. The dissociation constant of mant-ADP is identical to that for ADP, while that for mant-ATP is approximately threefold higher than that for ATP. The dissociation constant for mant-3'deoxyATP is approximately fivefold higher than that for 3'deoxyATP while derivatization of 2'deoxyATP does not affect affinity. The time-dependent binding of mant-ATP, mant-2'deoxyATP, and mant-ADP, measured using stopped-flow fluorescence spectroscopy, is best fit to three exponentials. The fast phase is ligand dependent, while the two slower phases are ligand independent. The slower phases are similar but not identical in rate, and have opposite fluorescence amplitudes. Both isomers of mant-ATP are equivalent substrates, as judged by reversed-phase chromatography, although the rate of phosphorylation is approximately 20-fold lower than the natural nucleotide. The kinetic data are consistent with a three-step binding mechanism in which initial association of the nucleotide derivatives produces a highly fluorescent complex. Either one or two conformational changes can occur after the formation of this binary species, but one of the isomerized forms must have low fluorescence compared to the initial binary complex. These data soundly attest to the structural plasticity within the kinase core that may be essential for catalysis. Overall, the mant nucleotides present a useful reporter system for gauging these conformational changes in light of the prevailing three-dimensional models for the enzyme.

Kinetics Processivity and the Direction of Motion of Ncd

The kinetic mechanism of the nonclaret disjunctional protein (Ncd) motor was investigated using the dimer termed MC1 (residues 209–700), which has been shown to exhibit negative-end directed motility (Chandra et al., 1993). The kinetic properties are similar to those of the monomeric Ncd motor domain (Pechatnikova and Taylor, 1997). The maximum steady-state ATPase activity of 1.5 s −1 is half as large as for the monomeric motor. Dissociation constants in the presence of nucleotides showed the same trend but with approximately a two-fold decrease in the values: K d values are 1.0 μM for ADP-AlF 4, 1.1 μM for ATP γS, 1.5 μM for ATP, 3 μM for ADP, and 10 μM for ADP-vanadate (in 25 mM NaCl, 22°C). The apparent second-order rate constants for the binding of ATP and ADP to the microtubule-motor complex (MtMC1) are 2 μM −1 s −1. Based on measurements at high microtubule concentrations the kinetic steps were fitted to the scheme, {formula not available us MathML} where N refers to one head of the dimer and T, D, and P stand for ATP, ADP, and inorganic phosphate. k 1 and k −4 are the first-order rate constants of the transition induced by the binding of mant ATP and mant ADP respectively. ADP release is the main rate-limiting step in the MtMC1 mechanism. The binding of the MC1–mant ADP complex to microtubules released less than half of the mant ADP (alternating site reactivity). The second mant ADP is only released by the binding of nucleotides that dissociate the MtMC1 complex (ATP and ADP but not AMPPNP). The apparent rate constant for dissociation of the second mant ADP is four times smaller than the first and much smaller than the rate of dissociation of MtMC1 by ATP or ADP. These results are explained by a model in which MC1·ADP is first dissociated from the microtubule by ATP, followed by rebinding to the microtubule by the ADP-containing head. Ncd may follow a different reaction pathway than does kinesin, but the differences in rate constants do not explain the opposite direction of motion. The kinetic evidence and the high ratio of motile velocity to ATPase support a nonprocessive, low duty cycle mechanism for the Ncd motor.

Flavonoids: a class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein.

A hexahistidine-tagged C-terminal nucleotide-binding domain (H6-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated to fluorescence resonance-energy transfer. H6-NBD2 also bound all flavonoids known to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells, with similar affinities and relative efficiencies. Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4'-methoxy 3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of H6-NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by preincubation with ATP, or partly displaced upon ATP addition. Interestingly, kaempferide was also able to partly prevent the binding of the antiprogestin RU 486 to a hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, which occurred at the ATP site and extended to the vicinal steroid-interacting hydrophobic region, was completely prevented or displaced by kaempferide. All results indicate that flavonoids constitute a new class of modulators with bifunctional interactions at vicinal ATP-binding site and steroid-interacting region within a cytosolic domain of P-glycoprotein.

Pathway of ATP hydrolysis by monomeric and dimeric kinesin.

The ATPase mechanism for a monomeric Drosophila kinesin construct, K341, was determined by pre-steady-state kinetic methods and compared to dimeric kinesin, K401. We directly measured the kinetics of binding mantATP (a fluorescent ATP analog) to the microtubule K341 complex, the dissociation of K341 from the microtubule, and release of phosphate and ADP from K341. Measurements of phosphate release kinetics at low salt concentration show that K341 hydrolyzes 18 molecules of ATP per kinesin monomer prior to release from the microtubule. At a higher salt concentration the amplitude of the pre-steady-state burst of phosphate release was reduced to 8 molecules per kinesin monomer. The maximum rate of dissociation of K341 from the microtubule following the addition of ATP was 22 s-1. The rate of mantADP release from the M.K341.mantADP complex increased as a function of tubulin concentration with a second-order rate constant of 11 microM-1 s-1 for K341 binding to the microtubule and reached a maximum rate of mantADP release of 303 s-1. ADP release kinetics were also determined by monitoring the binding of mantATP to K341.ADP and K401.ADP after mixing with microtubules. We show that monomeric kinesin remains associated with the microtubule through multiple rounds of ATP hydrolysis. This apparent processivity implies that one of the functions of the cooperative interaction between the two kinesin heads in dimeric kinesin is for the reactions occurring on one kinesin head to facilitate the release of the adjacent head from the microtubule.

Binding of steroid modulators to recombinant cytosolic domain from mouse P-glycoprotein in close proximity to the ATP site.

We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395-581 of mouse mdr1 P-glycoprotein bound fluorescent 2'(3')-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J. -C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem. 271, 11652-11658]. The present work shows that a longer 371-705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Delta6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 microM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.