Force generation in single conventional actomyosin complexes under high dynamic load

The mechanical load borne by a molecular motor affects its force, sliding distance, and its rate of energy transduction. The control of ATPase activity by the mechanical load on a muscle tunes its efficiency to the immediate task, increasing ATP hydrolysis as the power output increases at forces less than isometric (the Fenn effect) and suppressing ATP hydrolysis when the force is greater than isometric. In this work, we used a novel 'isometric' optical clamp to study the mechanics of myosin II molecules to detect the reaction steps that depend on the dynamic properties of the load. An actin filament suspended between two beads and held in separate optical traps is brought close to a surface that is sparsely coated with motor proteins on pedestals of silica beads. A feedback system increases the effective stiffness of the actin by clamping the force on one of the beads and moving the other bead electrooptically. Forces measured during actomyosin interactions are increased at higher effective stiffness. The results indicate that single myosin molecules transduce energy nearly as efficiently as whole muscle and that the mechanical control of the ATP hydrolysis rate is in part exerted by reversal of the force-generating actomyosin transition under high load without net utilization of ATP.     

Direct observation of chemo-mechanical coupling in DnaK by single-molecule force experiments

Protein allostery requires a communication channel for functional regulation between distal sites within a protein. In the molecular chaperone Hsp70, a two-domain enzyme, the ATP/ADP status of an N-terminal nucleotide-binding domain regulates the substrate affinity of a C-terminal substrate-binding domain. Recently available three-dimensional structures of Hsp70 in ATP/ADP states have provided deep insights into molecular pathways of allosteric signals. However, direct mechanical probing of long-range allosteric coupling between the ATP hydrolysis step and domain states is missing. Using laser optical tweezers, we examined the mechanical properties of a truncated two-domain DnaK(1–552ye) in apo/ADP/ATP- and peptide-bound states. We find that in the apo and ADP states, DnaK domains are mechanically stable and rigid. However, in the ATP state, substrate-binding domain (SBD)¹ye is mechanically destabilized as the result of interdomain docking followed by the unfolding of the α-helical lid. By observing the folding state of the SBD, we could observe the continuous ATP/ADP cycling of the enzyme in real time with a single molecule. The SBD lid closure is strictly coupled to the chemical steps of the ATP hydrolysis cycle even in the presence of peptide substrate.     

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.     

ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase

The enzyme F1-adenosine triphosphatase (ATPase) is a molecular motor that converts the chemical energy stored in the molecule adenosine triphosphate (ATP) into mechanical rotation of its gamma-subunit. During steady-state catalysis, the three catalytic sites of F1 operate in a cooperative fashion such that at every instant each site is in a different conformation corresponding to a different stage along the catalytic cycle. Notwithstanding a large amount of biochemical and, recently, structural data, we still lack an understanding of how ATP hydrolysis in F1 is coupled to mechanical motion and how the catalytic sites achieve cooperativity during rotatory catalysis. In this publication, we report combined quantum mechanical/molecular mechanical simulations of ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase. Our simulations reveal a dramatic change in the reaction energetics from strongly endothermic in betaTP to approximately equienergetic in betaDP. The simulations identify the responsible protein residues, the arginine finger alphaR373 being the most important one. Similar to our earlier study of betaTP, we find a multicenter proton relay mechanism to be the energetically most favorable hydrolysis pathway. The results elucidate how cooperativity between catalytic sites might be achieved by this remarkable molecular motor.     

ATP-synthase--intracellular molecular turbine

Literature data on molecular and functional organization of ATP synthase as turbine-like rotary motor are reviewed. These data now provide the first experimental evidence that ATP synthases from different sources are able to reversibly couple the proton-motive force (proton electrochemical potential) with the synthesis/hydrolysis of ATP by the mechanical rotation of the rotor component of enzyme relative to the its stator part.     

Ser203在大鼠脑驱动蛋白水解ATP中重要作用的晶体学分析

鼠脑驱动蛋白（rat brain kinesin）是一种利用水解ATP所释放的能量在微管束上高速并且连续性运动的常规驱动蛋白. 它在神经突触的物质运输中起着重要作用. 研究驱动蛋白是如何将ATP中储藏的化学能转化为机械动能是理解其运动机能的重要课题. 本课题获得了鼠脑驱动蛋白单体与ATP结构类似物AMPPCP形成的复合物晶体结构. 将这个晶体结构与鼠脑驱动蛋白单体-另一种ATP结构类似物AMPPNP形成的复合物晶体结构以及鼠脑驱动蛋白单体-ATP水解产物ADP形成的复合物晶体结构进行相互比较,揭示了活性中心的开关区域I中丝氨酸203可能作为质子的供体,加速了ATP中gamma-磷酸和beta-磷酸的断裂,从而导致ATP的水解.     

Regulation of the Pi-ATP exchange and hydrolytic reactions in F0-F1 reconstituted liposomes

The regulation of ATP hydrolysis and Pi-ATP exchange reactions by ATP, ADP, Mg2+, and phosphate was studied in liposomes containing F0-F1 obtained from bovine heart submitochondrial particles by solubilization with lauryl dimethylamino oxide as described previously (Dreyfus, G., Celis, H., and Ramirez, J. (1984) Anal. Biochem. 142, 215-220). A simultaneous analysis of ATP hydrolysis and the Pi-ATP exchange reactions showed that the ratio of hydrolysis/exchange is close to one when the ATP concentration is in the lower micromolar range. In this preparation ADP stimulates the Pi-ATP exchange reaction and depresses ATP hydrolysis. The exchange reaction is almost abolished when ADP is removed from the medium by an ATP-regenerating system. Mg2+ in millimolar concentrations stimulates Pi-ATP exchange, and at the same time decreases ATP hydrolysis; accordingly, the hydrolysis/exchange ratio depends on the concentration of Mg2+. Inorganic phosphate also controls this ratio, a lower ratio being observed at high phosphate concentrations. The Pi-ATP exchange reaction, but not ATP hydrolysis, depends on the concentration of medium phosphate. These results indicate that the kinetic characteristics of this F0-F1 preparation are modified by Mg2+, ATP, and phosphate.     

Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli

RecA is essential for recombination, DNA repair and SOS induction in Escherichia coli . ATP hydrolysis is known to be important for RecA's roles in recombination and DNA repair. In vitro reactions modelling SOS induction minimally require ssDNA and non-hydrolyzable ATP analogues. This predicts that ATP hydrolysis will not be required for SOS induction in vivo . The requirement of ATP binding and hydrolysis for SOS induction in vivo is tested here through the study of recA4159 (K72A) and recA2201 (K72R). RecA4159 is thought to have reduced affinity for ATP. RecA2201 binds, but does not hydrolyse ATP. Neither mutant was able to induce SOS expression after UV irradiation. RecA2201, unlike RecA4159, could form filaments on DNA and storage structures as measured with RecA–GFP. RecA2201 was able to form hybrid filaments and storage structures and was either recessive or dominant to RecA⁺, depending on the ratio of the two proteins. RecA4159 was unable to enter RecA⁺ filaments on DNA or storage structures and was recessive to RecA⁺. It is concluded that ATP hydrolysis is essential for SOS induction. It is proposed that ATP binding is essential for storage structure formation and ability to interact with other RecA proteins in a filament.     

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation

F(1)-ATPase is a rotary molecular motor in which unidirectional rotation of the central gamma subunit is powered by ATP hydrolysis in three catalytic sites arranged 120 degrees apart around gamma. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of gamma, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an approximately 80 degrees rotation of gamma; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further approximately 40 degrees rotation.     

An ATP hydrolysis sensor in the DNA packaging motor from bacteriophage T4 suggests an inchworm-type translocation mechanism

Tailed bacteriophages and large eukaryotic viruses employ powerful molecular motors to translocate dsDNA into a preassembled capsid shell. The phage T4 motor is composed of a dodecameric portal and small and large terminase subunits assembled at the special head-tail connector vertex of the prohead. The motor pumps DNA through the portal channel, utilizing ATP hydrolysis energy provided by an ATPase present in the large terminase subunit. We report that the ATPase motors of terminases, helicases, translocating restriction enzymes, and protein translocases possess a common coupling motif (C-motif). Mutations in the phage T4 terminase C-motif lead to loss of stimulated ATPase and DNA translocation activities. Surprisingly, the mutants can catalyze at least one ATP hydrolysis event but are unable to turn over and reset the motor. This is the first report of a catalytic block in translocating ATPase motor after ATP hydrolysis occurred. We suggest that the C-motif is an ATP hydrolysis sensor, linking product release to mechanical motion. A novel terminase-driven mechanism is proposed for translocation of dsDNA in viruses.     

Kinetics of tryptophan fluorescence enhancement in myofibrils during ATP hydrolysis

The mechanism of ATP hydrolysis in myofibrils can be studied by following the time course of tryptophan fluorescence. Stoichiometric quantities of ATP produce an enhancement of the tryptophan fluorescence in stirred suspensions of rabbit psoas myofibrils at pCa greater than 7. Approximately 1 mol of ATP/myosin head is required to obtain the maximum fluorescence enhancement of 4-6%. Upon the addition of quantities of ATP greater than 1 mol/mol of myosin head, the fluorescence rapidly increases to a steady state, which lasts for a period that is proportional to the amount of ATP added. The fluorescence then decays to the initial level with a half-time of approximately 40 s at 20 degrees C. Hydrolysis of [gamma-32P]ATP at pCa greater than 7 in myofibrils has an initial burst of approximately 0.7 mol/mol of myosin head that is followed by a constant rate of hydrolysis. The duration of the steady state hydrolysis is identical to the duration of the enhancement of tryptophan fluorescence. A lower limit of 5 X 10(5) M-1 S-1 was obtained for the second order rate constant of the fluorescence enhancement by ATP. At pCa of 4, the duration of the fluorescence enhancement is one-tenth to one-twentieth as long as at pCa greater than 7; this is consistent with the increased steady state rate of ATP hydrolysis at higher calcium concentrations. The time course of the fluorescence enhancement observed in myofibrils during ATP hydrolysis is qualitatively and quantitatively similar to that observed with actomyosin-S1 in solution. These results suggest that the kinetic mechanism of ATP hydrolysis that has been well established by studies of actomyosin-S1 in solution also occurs in myofibrils.     

Mechanism of loading the Escherichia coli DNA polymerase III sliding clamp: I. Two distinct activities for individual ATP sites in the gamma complex

The Escherichia coli DNA polymerase III gamma complex loads the beta clamp onto DNA, and the clamp tethers the core polymerase to DNA to increase the processivity of synthesis. ATP binding and hydrolysis promote conformational changes within the gamma complex that modulate its affinity for the clamp and DNA, allowing it to accomplish the mechanical task of assembling clamps on DNA. This is the first of two reports (Snyder, A. K., Williams, C. R., Johnson, A., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4386-4393) addressing the question of how ATP binding and hydrolysis modulate specific interactions with DNA and beta. Pre-steady-state rates of ATP hydrolysis were slower when reactions were initiated by addition of ATP than when the gamma complex was equilibrated with ATP and were limited by the rate of an intramolecular reaction, possibly ATP-induced conformational changes. Kinetic modeling of assays in which the gamma complex was incubated with ATP for different periods of time prior to adding DNA to trigger hydrolysis suggests a mechanism in which a relatively slow conformational change step (kforward = 6.5 s(-1)) produces a species of the gamma complex that is activated for DNA (and beta) binding. In the absence of beta, 2 of the 3 molecules of ATP are hydrolyzed rapidly prior to releasing DNA, and the 3rd molecule is hydrolyzed slowly. In the presence of beta, all 3 molecules of ATP are hydrolyzed rapidly. These results suggest that hydrolysis of 2 molecules of ATP may be coupled to conformational changes that reduce interactions with DNA, whereas hydrolysis of the 3rd is coupled to changes that result in release of beta.     

Secondary and tertiary structure changes of reconstituted LmrA induced by nucleotide binding or hydrolysis. A fourier transform attenuated total reflection infrared spectroscopy and tryptophan fluorescence quenching analysis

LmrA, a membrane protein of Lactococcus lactis, extrudes amphiphilic compounds from the inner leaflet of the cytoplasmic membrane, using energy derived from ATP hydrolysis. A combination of total reflection Fourier transform infrared spectroscopy, (2)H/H exchange, and fluorescence quenching experiments was used to investigate the effect of nucleotide binding and/or hydrolysis on the structure of LmrA reconstituted into proteoliposomes. These measurements allowed us to describe secondary structure changes of LmrA during the catalytic cycle. The structure of LmrA is enriched in beta-sheet after ATP binding, and the protein recovers its initial secondary structure after ATP hydrolysis, when P(i) has been released. (2)H/H exchange and fluorescence quenching studies indicate that the protein undergoes two distinct tertiary structure changes during the hydrolysis process. Indeed, the protein alone is poorly accessible to the aqueous medium but adopts a more accessible conformation when ATP hydrolysis takes place. After ATP hydrolysis, but when P(i) is still associated with the protein, the accessibility is intermediate between these two states.     

Matters Arising

Abstract: The possibility that neurofilaments could be involved in the transduction of chemical and mechanical energy in axons led us to investigate whether neurofilament proteins can hydrolyze ATP. We fractionated neurofilaments from rabbit spinal cord and found that preparations highly enriched for neurofilament proteins hydrolyzed ATP at a substantial rate (as high as 0.4 μmol/min/mg protein). However, the ATPase activity was neither inhibited by anti-neurofilament antibody, nor was it precipitated by the antibody under circumstances that precipitated most of the neurofilament polypeptides. We conclude that neurofilament proteins do not hydrolyze ATP at a significant rate under the conditions of our assay; if hydrolysis of ATP is a physiological function of neurofilaments, additional factors are required.     

Asp-52 in Combination with Asp-398 Plays a Critical Role in ATP Hydrolysis of Chaperonin GroEL

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists protein folding with the aid of GroES and ATP. Asp-398 in GroEL is known as one of the critical residues on ATP hydrolysis because GroEL(D398A) mutant is deficient in ATP hydrolysis (<2% of the wild type) but not in ATP binding. In the archaeal Group II chaperonin, another aspartate residue, Asp-52 in the corresponding E. coli GroEL, in addition to Asp-398 is also important for ATP hydrolysis. We investigated the role of Asp-52 in GroEL and found that ATPase activity of GroEL(D52A) and GroEL(D52A/D398A) mutants was ∼20% and <0.01% of wild-type GroEL, respectively, indicating that Asp-52 in E. coli GroEL is also involved in the ATP hydrolysis. GroEL(D52A/D398A) formed a symmetric football-shaped GroEL-GroES complex in the presence of ATP, again confirming the importance of the symmetric complex during the GroEL ATPase cycle. Notably, the symmetric complex of GroEL(D52A/D398A) was extremely stable, with a half-time of ∼150 h (∼6 days), providing a good model to characterize the football-shaped complex.     

Analysis of the Cooperative ATPase Cycle of the AAA+ Chaperone ClpB from Thermus thermophilus by Using Ordered Heterohexamers with an Alternating Subunit Arrangement

The ClpB/Hsp104 chaperone solubilizes and reactivates protein aggregates in cooperation with DnaK/Hsp70 and its cofactors. The ClpB/Hsp104 protomer has two AAA+ modules, AAA-1 and AAA-2, and forms a homohexamer. In the hexamer, these modules form a two-tiered ring in which each tier consists of homotypic AAA+ modules. By ATP binding and its hydrolysis at these AAA+ modules, ClpB/Hsp104 exerts the mechanical power required for protein disaggregation. Although ATPase cycle of this chaperone has been studied by several groups, an integrated understanding of this cycle has not been obtained because of the complexity of the mechanism and differences between species. To improve our understanding of the ATPase cycle, we prepared many ordered heterohexamers of ClpB from Thermus thermophilus, in which two subunits having different mutations were cross-linked to each other and arranged alternately and measured their nucleotide binding, ATP hydrolysis, and disaggregation abilities. The results indicated that the ATPase cycle of ClpB proceeded as follows: (i) the 12 AAA+ modules randomly bound ATP, (ii) the binding of four or more ATP to one AAA+ ring was sensed by a conserved Arg residue and converted another AAA+ ring into the ATPase-active form, and (iii) ATP hydrolysis occurred cooperatively in each ring. We also found that cooperative ATP hydrolysis in at least one ring was needed for the disaggregation activity of ClpB.     

Myosin motor domain lever arm rotation is coupled to ATP hydrolysis

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.     

Systematic study of the functions for the residues around the nucleotide pocket in simian virus 40 AAA+ hexameric helicase

The high-resolution structural data for simian virus 40 large-T-antigen helicase revealed a set of nine residues bound to ATP/ADP directly or indirectly. The functional role of each of these residues in ATP hydrolysis and also the helicase function of this AAA+ (ATPases associated with various cellular activities) molecular motor are unclear. Here, we report our mutational analysis of each of these residues to examine their functionality in oligomerization, DNA binding, ATP hydrolysis, and double-stranded DNA (dsDNA) unwinding. All mutants were capable of oligomerization in the presence of ATP and could bind single-stranded DNA and dsDNA. ATP hydrolysis was substantially reduced for proteins with mutations of residues making direct contact with the gamma-phosphate of ATP or the apical water molecule. A potentially noncanonical "arginine finger" residue, K418, is critical for ATP hydrolysis and helicase function, suggesting a new type of arginine finger role by a lysine in the stabilization of the transition state during ATP hydrolysis. Interestingly, our mutational data suggest that the positive- and negative-charge interactions in the uniquely observed residue pairs, R498/D499 and R540/D502, in large-T-antigen helicase are critically involved in the transfer of energy of ATP binding/hydrolysis to DNA unwinding.     

PcrA helicase, a prototype ATP-driven molecular motor

Despite extensive studies, the mechanisms underlying molecular motor function are still poorly understood. Key to the mechanisms is the coupling of ATP hydrolysis to conformational changes of the motor protein. To investigate this coupling, we have conducted combined quantum mechanical/molecular mechanical simulations of PcrA helicase, a strikingly simple motor that translocates unidirectionally along single-stranded DNA (ssDNA). Our results reveal a close similarity in catalytic site structure and reaction pathway to those of F1-ATPase, and these similarities include a proton relay mechanism important for efficient ATP hydrolysis and an "arginine finger" residue that is key to the coupling of the chemical reaction to protein conformational changes. By means of in silico mutation studies, we identified the residue Q254 as being crucial for the coupling of ssDNA translocation to the actual catalytic event. Based on the present result for PcrA helicase and previous findings for F1-ATPase, we propose a general mechanism of ATP-driven molecular motor function.