ATPase Site Research Articles

Identification and Biochemical Characterization of Halisulfate 3 and Suvanine as Novel Inhibitors of Hepatitis C Virus NS3 Helicase from a Marine Sponge

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

Crystal and EM Structures Reveal a Mechanism for Long-Range Allosteric Communication in the Yeast Dynein Motor Domain

Dynein, a member of the AAA family of ATPases, “walks” toward the minus end of microtubules using energy from ATP hydrolysis. Recent atomic structures of the dynein motor domain with no nucleotide (apo; yeast) or ADP (Dictyostelium) in the main hydrolytic site (AAA1) have begun to yield insights into how this fascinatingly complex machine may work. Here, we have solved the crystal (3.5 A) of the yeast dynein motor domain with a nonhydrolyzable nucleotide (AMPPNP) bound at AAA1. By comparison to the prior apo structures of yeast dynein, the new structure reveals that AMPPNP causes a large conformational change of AAA domains in the dynein ring in which AAA2 moves closer to AAA1. The linker, the proposed mechanical element of dynein, remains largely in a similar conformation with its N-terminal docked onto AAA5, but new interactions are made with AAA2. Further EM analysis of dynein bound to several ATP analogs showed that the linker remains docked on the ring in all states, with a small conformational change observed when bound to the analog ATP-vanadate. Mutagenesis studies show that disruption of a single salt-bridge between the linker and AAA2 blocks microtubule stimulation of AAA1 ATPase activity and nucleotide-induced changes in microtubule binding affinity. Collectively, our data reveal a new nucleotide triphosphate bound state of the dynein motor domain and suggest that the linker serves not only in dynein mechanics but also plays a critical role in the long range allosteric communication between dynein's main ATPase site and the microtubule binding domain.

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.

Michaelis–Menten at 100 and allosterism at 50: driving molecular motors in a hailstorm with noisy ATPase engines and allosteric transmission

Cytoskeletal motor proteins move on filamentous tracks by converting input chemical energy that they derive by catalyzing the hydrolysis of ATP. The ATPase site is the analogue of an engine and hydrolysis of ATP is the analogue of burning of chemical fuel. Moreover, the functional role of a segment of the motor is analogous to that of the transmission system of an automobile, which consists of a shaft, gear, clutch, etc. The operation of the engine is intrinsically 'noisy' and the motor faces a molecular 'hailstorm' in the aqueous medium. In this commemorative review, we celebrate the centenary of Michaelis and Menten's landmark paper of 1913 and the golden jubilee of Monod and colleagues classic paper of 1963 by highlighting their relevance with respect to explaining the operational mechanisms of the engine and the transmission system, respectively, of cytoskeletal motors.

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.

E. coli ClpA Catalyzed Polypeptide Translocation Is Allosterically Controlled by the Protease ClpP

There are five known ATP-dependent proteases in Escherichia coli (Lon, ClpAP, ClpXP, HslUV, and the membrane-associated FtsH) that catalyze the removal of both misfolded and properly folded proteins in cellular protein quality control pathways. Hexameric ClpA rings associate with one or both faces of the cylindrically shaped tetradecameric ClpP protease. ClpA catalyzes unfolding and translocation of polypeptide substrates into the proteolytic core of ClpP for degradation through repeated cycles of ATP binding and hydrolysis at two nucleotide binding domains on each ClpA monomer. We previously reported a molecular mechanism for ClpA catalyzed polypeptide translocation in the absence of ClpP, including elementary rate constants, overall rate, and the kinetic step size. However, the potential allosteric effect of ClpP on the mechanism of ClpA catalyzed translocation remains unclear. Using single-turnover fluorescence stopped-flow methods, here we report that ClpA, when associated with ClpP, translocates polypeptide with an overall rate of ~35 aa s−1 and, on average, traverses ~5 aa between two rate-limiting steps with reduced cooperativity between ATP binding sites in the hexameric ring. This is in direct contrast to our previously reported observation that, in the absence of ClpP, ClpA translocates polypeptide substrates with a maximum translocation rate of ~20 aa s−1 with cooperativity between ATPase sites. Our results demonstrate that ClpP allosterically impacts the polypeptide translocation activity of ClpA by reducing the cooperativity between ATP binding sites.

Direct real-time detection of the actin-activated power stroke within the myosin catalytic domain

We have used transient kinetics, nanosecond time-resolved fluorescence resonance energy transfer (FRET), and kinetics simulations to resolve a structural transition in the Dictyostelium myosin II relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with the force-transducing rotation of the myosin light-chain domain. Previous research in the absence of actin showed that ATP binding to myosin induces a dynamic equilibrium between a bent prepower stroke state of the relay helix and a straight postpower stroke state, which dominates in the absence of ATP or when ADP is bound. We now ask whether actin binding reverses this transition and if so, how this reversal is coordinated with actin-activated phosphate release. We labeled a Cys-lite Dictyostelium myosin II motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We then performed transient time-resolved FRET following stopped-flow mixing of actin with labeled myosin, preincubated with ATP. We determined the kinetics of actin-activated phosphate release, using fluorescent phosphate-binding protein. The results show that actin binding to the myosin.ADP.P complex straightens the relay helix before phosphate dissociation. This actin-activated relay helix straightening is reversible, but phosphate irreversibly dissociates from the postpower stroke state, preventing reversal of the power stroke. Thus, relay helix straightening gates phosphate dissociation, whereas phosphate dissociation provides the thermodynamic driving force underlying force production.

A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B 12 utilization

In all kingdoms of life, ATP binding cassette (ABC) transporters are essential to many cellular functions. In this large superfamily of proteins, two catalytic sites hydrolyze ATP to power uphill substrate translocation. A central question in the field concerns the relationship between the two ATPase catalytic sites: Are the sites independent of one another? Are both needed for function? Do they function cooperatively? These issues have been resolved for type I ABC transporters but never for a type II ABC transporter. The many mechanistic differences between type I and type II ABC transporters raise the question whether in respect to ATP hydrolysis the two subtypes are similar or different. We have addressed this question by studying the Escherichia coli vitamin B12 type II ABC transporter BtuCD. We have constructed and purified a series of BtuCD variants where both, one, or none of the ATPase sites were rendered inactive by mutation. We find that, in a membrane environment, the ATPase sites of BtuCD are highly cooperative with a Hill coefficient of 2. We also find that, when one of the ATPase sites is inactive, ATP hydrolysis and vitamin B12 transport by BtuCD is reduced by 95%. These exact features are also shared by the archetypical type I maltose ABC transporter. Remarkably, mutants that have lost 95% of their ATPase and transport capabilities still retain the ability to fully use vitamin B12 in vivo. The results demonstrate that, despite the many differences between type I and type II ABC transporters, the fundamental mechanism of ATP hydrolysis remains conserved.

An Expanding Range of Functions for the Copper Chaperone/Antioxidant Protein Atox1

Antioxidant protein 1 (Atox1 in human cells) is a copper chaperone for the copper export pathway with an essential role in cellular copper distribution. In vitro, Atox1 binds and transfers copper to the copper-transporting ATPases, stimulating their catalytic activity. Inactivation of Atox1 in cells inhibits maturation of secreted cuproenzymes as well as copper export from cells. Accumulating data suggest that cellular functions of Atox1 are not limited to its copper-trafficking role and may include storage of labile copper, modulation of transcription, and antioxidant defense. The conserved metal binding site of Atox1, CxGC, differs from the metal-binding sites of copper-transporting ATPases and has a physiologically relevant redox potential that equilibrates with the GSH:GSSG pair. Tight relationship appears to exist between intracellular copper levels and glutathione (GSH) homeostasis. The biochemical properties of Atox1 place it at the intersection of cellular networks that regulate copper distribution and cellular redox balance. Mechanisms through which Atox1 facilitates copper export and contributes to oxidative defense are not fully understood. The current picture of cellular redox homeostasis and copper physiology will be enhanced by further mechanistic studies of functional interactions between the GSH:GSSG pair and copper-trafficking machinery.

The Naphthoquinone Diospyrin Is an Inhibitor of DNA Gyrase with a Novel Mechanism of Action

Tuberculosis and other bacterial diseases represent a significant threat to human health. The DNA topoisomerases are excellent targets for chemotherapy, and DNA gyrase in particular is a well-validated target for antibacterial agents. Naphthoquinones (e.g. diospyrin and 7-methyljuglone) have been shown to have therapeutic potential, particularly against Mycobacterium tuberculosis. We have found that these compounds are inhibitors of the supercoiling reaction catalyzed by M. tuberculosis gyrase and other gyrases. Our evidence strongly suggests that the compounds bind to the N-terminal domain of GyrB, which contains the ATPase active site, but are not competitive inhibitors of the ATPase reaction. We propose that naphthoquinones bind to GyrB at a novel site close to the ATPase site. This novel mode of action could be exploited to develop new antibacterial agents.

Coordination of Actin-Activated Phosphate Release and the Myosin Power-Stroke Detected by Transient Time-Resolved FRET

We have used transient kinetics and nanosecond time-resolved FRET to resolve a structural transition in the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in myosin, coupling biochemical changes in the ATPase site with force-transducing rotation of the myosin light chain domain. Previous research in the absence of actin used optical and EPR spectroscopy to demonstrate that the relay helix of Dictyostelium Discoideum myosin II exists in dynamic equilibrium between a bent pre-power-stroke state, stabilized by ATP, and a straight post-power-stroke state that predominates when ADP or no nucleotide is bound. Kinetics studies showed that ATP binding induces a recovery stroke in which this equilibrium is shifted toward the bent state. In the present study, we ask whether actin binding to the myosin.ADP.Pi complex reverses this transition and if so, how actin-activated helix straightening is coordinated with biochemical transitions of the myosin ATPase cycle. We labeled a Cys-lite Dictyostelium myosin motor domain with donor and acceptor probes at two engineered Cys residues designed to detect relay helix bending. We measured time-resolved FRET following stopped-flow mixing of myosin (in the presence of ATP) with actin. These experiments allowed us to measure the structural kinetics of the relay helix during the weak-to-strong actin-binding transition. The results clearly show that actin binding straightens the relay helix in the presence of ATP. The helix straightens prior to ADP dissociation and full formation of the strong binding actomyosin interface. Measurements of actin-activated phosphate release demonstrate that dissociation of the hydrolyzed gamma-phosphate is closely coupled to the structural state of the relay helix during the power-stroke.

Astrocytic and neuronal accumulation of elevated extracellular K+ with a 2/3 K+/Na+ flux ratio—consequences for energy metabolism, osmolarity and higher brain function

Brain excitation increases neuronal Na+ concentration by 2 major mechanisms: (i) Na+ influx caused by glutamatergic synaptic activity; and (ii) action-potential-mediated depolarization by Na+ influx followed by repolarizating K+ efflux, increasing extracellular K+ concentration. This review deals mainly with the latter and it concludes that clearance of extracellular K+ is initially mainly effectuated by Na+,K+-ATPase-mediated K+ uptake into astrocytes, at K+ concentrations above ~10 mM aided by uptake of Na+,K+ and 2 Cl− by the cotransporter NKCC1. Since operation of the astrocytic Na+,K+-ATPase requires K+-dependent glycogenolysis for stimulation of the intracellular ATPase site, it ceases after normalization of extracellular K+ concentration. This allows K+ release via the inward rectifying K+ channel Kir4.1, perhaps after trans-astrocytic connexin- and/or pannexin-mediated K+ transfer, which would be a key candidate for determination by synchronization-based computational analysis and may have signaling effects. Spatially dispersed K+ release would have little effect on extracellular K+ concentration and allow K+ accumulation by the less powerful neuronal Na+,K+-ATPase, which is not stimulated by increases in extracellular K+. Since the Na+,K+-ATPase exchanges 3 Na+ with 2 K+, it creates extracellular hypertonicity and cell shrinkage. Hypertonicity stimulates NKCC1, which, aided by β-adrenergic stimulation of the Na+,K+-ATPase, causes regulatory volume increase, furosemide-inhibited undershoot in [K+]e and perhaps facilitation of the termination of slow neuronal hyperpolarization (sAHP), with behavioral consequences. The ion transport processes involved minimize ionic disequilibria caused by the asymmetric Na+,K+-ATPase fluxes.

Mn2+–Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance

Pulsed electron paramagnetic resonance at the microwave K(a) band (~30 GHz) was used to study the coordination of adenosine nucleotides to Mn(2+) at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electron-nuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mn·nucleotide complexes. Therefore, to directly detect binding of the Mn·nucleotide to myosin, we used nonhydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electron-electron double resonance to detect spin probe-manganese dipolar interaction. We found that under substoichiometric conditions, both Mn·AMPPNP and Mn·ADP·AlF(4) form a complex with myosin, and Mn·ADP does not form such a complex. This correlates well with the biological dissociation of Mg·ADP from myosin after the hydrolysis of ATP. The analysis of (31)P ENDOR spectra reveals that in Mn·AMPPNP, Mn·ATP, and Mn·ADP at myosin or in solution, the nucleotide is coordinated to Mn(2+) by two phosphate groups, whereas in Mn·ADP·AlF(4), only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of Mn·ADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ions is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metal-protein binding when such binding does not provide reliable spectroscopic signatures.

Lis1 Acts as a “Clutch” between the ATPase and Microtubule-Binding Domains of the Dynein Motor

SummaryThe lissencephaly protein Lis1 has been reported to regulate the mechanical behavior of cytoplasmic dynein, the primary minus-end-directed microtubule motor. However, the regulatory mechanism remains poorly understood. Here, we address this issue using purified proteins from Saccharomyces cerevisiae and a combination of techniques, including single-molecule imaging and single-particle electron microscopy. We show that rather than binding to the main ATPase site within dynein's AAA+ ring or its microtubule-binding stalk directly, Lis1 engages the interface between these elements. Lis1 causes individual dynein motors to remain attached to microtubules for extended periods, even during cycles of ATP hydrolysis that would canonically induce detachment. Thus, Lis1 operates like a “clutch” that prevents dynein's ATPase domain from transmitting a detachment signal to its track-binding domain. We discuss how these findings provide a conserved mechanism for dynein functions in living cells that require prolonged microtubule attachments.

ATPase Site Architecture Is Required for Self-Assembly and Remodeling Activity of a Hexameric AAA+ Transcriptional Activator

SummaryAAA+ proteins (ATPases associated with various cellular activities) are oligomeric ATPases that use ATP hydrolysis to remodel their substrates. By similarity with GTPases, a dynamic organization of the nucleotide-binding pockets between ATPase protomers is proposed to regulate functionality. Using the transcription activator PspF as an AAA+ model, we investigated contributions of conserved residues for roles in ATP hydrolysis and intersubunit communication. We determined the R-finger residue and revealed that it resides in a conserved “R-hand” motif (RxDxxxR) needed for its “trans-acting” activity. Further, a divergent Walker A glutamic acid residue acts synergistically with a tyrosine residue to function in ADP-dependent subunit-subunit coordination, forming the “ADP-switch” motif. Another glutamic acid controls hexamer formation in the presence of nucleotides. Together, these results lead to a “residue-nucleotide” interaction map upon which to base AAA+ core regulation.

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2-7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2-7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2-7 spontaneously adopts a left-handed, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high- and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2-7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2-7 systems.

Biochemical Analysis of the Human Mismatch Repair Proteins hMutSα MSH2G674A-MSH6 and MSH2-MSH6T1219D

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.

Actin Straightens the Myosin Relay Helix during the Powerstroke

We have used transient time-resolved FRET, (TR)2FRET, to resolve the structural kinetics of the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in all myosin isoforms coupling structural changes in the ATPase site with rotation of the light chain binding domain and force generation. Previous work demonstrated that the relay helix of the myosin II motor domain from Dictyostelium Discoideum exists in dynamic equilibrium between a bent pre-powerstroke state stabilized by ATP, and a straight post-powerstroke state which predominates in the presence of ADP or when the nucleotide binding site is empty. We predict that actin binding to the myosin.ADP.Pi complex will reverse helix bending. This transition should parallel the myosin power-stroke. To test this hypothesis, a Cys-lite Dictyostelium myosin motor domain was labeled at two introduced Cys residues shown to detect relay helix bending using DEER, TR-FRET and (TR)2FRET spectroscopy. We preformed a (TR)2FRET experiment measuring the structural distribution of the relay helix every millisecond after rapid mixing of myosin.ADP/Pi with actin. This experiment showed that actin binding straightens the bent helix. We characterized the actin dependence of helix straightening and then correlated this dependence with the kinetics of actin binding and phosphate dissociation from the myosin active site. Numerical simulations were used to model a system containing pre and post-powerstroke structural states, actin binding, hydrolysis and phosphate dissociation. We globally fit this model to the (TR)2FRET, actin binding, and phosphate dissociation transients. From this analysis, we are able to determine the timing of helix straightening with respect to actin binding and the critical step of phosphate dissociation which acts as the thermodynamic driving force for myosin's force generating powerstroke.

Incorporation of Photochromic Molecule into the Functional Loop L5 of Kinesin for the Purpose of Photo-Regulation

ATP driven molecular motor kinesin has several unique loops, which may determine the characteristic properties of kinesin. L5 is one of the unique loops locates in the vicinity of ATP binding site. We have previously demonstrated that the point mutations in the loop drastically affect ATPase activity. Moreover, it seems that the plus-end directed kinesins have longer L5 loop than that of minus-end directed kinesins. Therefore, the loop may determine the directionality of kinesin. Rice plant kinesin K16 has much shorter L5 than that of conventional kinesin. We have prepared the K16 mutant Q101C and H102C that have a single reactive cysteine reside in the L5. SH group reactive photochromic molecule composed of azobenzene derivative, N- (4-phenylazophenyl) maleimide (PAM) was incorporated into the cysteine residue in L5 to induce conformational change of L5 by ultraviolet (UV) and visible (VIS) light irradiation. The kinesin modified by PAM slightly altered the ATPase activity by UV and VIS light irradiation reversibly. We have also focused on the mitotic kinesin Eg5 that has an unusually elongated L5. It is known that the L5 of Eg5 has an important role to stabilize sterically its specific inhibitors (monastrol, STLC) that bind to the pocket near the ATPase site. We designed Eg5 mutants, E116C, E118C, D130C, A133C and Y125C to introduce photochromic molecule into the L5 of Eg5. The Effect of photoisomerization of azobenzene derivatives incorporated into L5 upon UV-VIS light irradiation for the inhibition of ATPase activity by monastrol or STLC was studied.

Prediction of inhibitory activities of Hsp90 inhibitors

Here, we report on the development of a novel methodology to aid in design of Hsp90 inhibitors, using molecular docking combined with artificial neural network (ANN) modelling. Inhibitors are first docked into the ATPase site of the Human Hsp90α crystal structures and the thermodynamic properties of the complexes together with various physical–chemical properties of the ligands are used as input to train a simple feed-forward, back propagation ANN, to predict the inhibitors’ pIC50s. For an objective test set of 60 known Hsp90 inhibitors for which there are no crystallographic data available, the trained ANN is shown to give pIC50s accurate to within ±1 log unit, and the predictions are sufficiently good as to allow the majority of the inhibitors to be ranked correctly according to their potency.