Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.     

Crystal structure of atypical cytoplasmic ABC-ATPase SufC from Thermus thermophilus HB8

SufC, a cytoplasmic ABC-ATPase, is one of the most conserved Suf proteins. SufC forms a stable complex with SufB and SufD, and the SufBCD complex interacts with other Suf proteins in the Fe-S cluster assembly. We have determined the crystal structure of SufC from Thermus thermophilus HB8 in nucleotide-free and ADP-Mg-bound states at 1.7A and 1.9A resolution, respectively. The overall architecture of the SufC structure is similar to other ABC ATPases structures, but there are several specific motifs in SufC. Three residues following the end of the Walker B motif form a novel 3(10) helix which is not observed in other ABC ATPases. Due to the novel 3(10) helix, a conserved glutamate residue involved in ATP hydrolysis is flipped out. Although this unusual conformation is unfavorable for ATP hydrolysis, salt-bridges formed by conserved residues and a strong hydrogen-bonding network around the novel 3(10) helix suggest that the novel 3(10) helix of SufC is a rigid conserved motif. Compared to other ABC-ATPase structures, a significant displacement occurs at a linker region between the ABC alpha/beta domain and the alpha-helical domain. The linker conformation is stabilized by a hydrophobic interaction between conserved residues around the Q loop. The molecular surfaces of SufC and the C-terminal helices of SufD (PDB code: 1VH4) suggest that the unusual linker conformation conserved among SufC proteins is probably suitable for interacting with SufB and SufD.     

The kinetic mechanism of the SufC ATPase: the cleavage step is accelerated by SufB

Protein products of the suf operon are involved in iron-sulfur metabolism. SufC is an ATPase that can interact with SufB in the absence of nucleotide. We have studied the transient kinetics of the SufC ATPase mechanism using the fluorescent ATP analogue, 2'(3')-O-N-methylanthraniloyl-ATP (mantATP). mantATP initially binds to SufC weakly. A conformational change of the SufC.mantATP complex then occurs followed by the very slow cleavage of mantATP to mantADP and the rapid release of Pi. In the presence of SufB, the cleavage step is accelerated and the release of mantADP is inhibited. Both of these effects promote the formation of a SufC.mantADP complex. In the absence and presence of SufB, mantADP remains more tightly bound to SufC than mantATP. These studies provide a basis for how the SufB and -C proteins interact in the processes involved in regulating iron-sulfur transfer.     

The Walker B motif of the second nucleotide-binding domain (NBD2) of CFTR plays a key role in ATPase activity by the NBD1-NBD2 heterodimer

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory 'R' domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1-NBD2 heterodimer.     

Mutational analysis of carbamyl phosphate synthetase. Substitution of Glu841 leads to loss of functional coupling between the two catalytic domains of the synthetase subunit.

The synthetase subunit of Escherichia coli carbamyl phosphate synthetase has two catalytic nucleotide-binding domains, one involved in the activation of HCO3- and the second in phosphorylation of carbamate. Here we show that a Glu841----Lys841 substitution in a putative ATP-binding domain located in the carboxyl half of the synthetase abolishes overall synthesis of carbamyl phosphate with either glutamine or NH3 as the nitrogen source. Measurements of partial activities indicate that while HCO3(-)-dependent ATP hydrolysis at saturating concentrations of substrate proceeds at higher than normal rates, ATP synthesis from ADP and carbamyl phosphate is nearly completely suppressed by the mutation. These results indicate Glu841 to be an essential residue for the phosphorylation of carbamate in the terminal step of the catalytic mechanism. The Lys841 substitution also affects the kinetic properties of the HCO3- activation site. Both kcat and Km for ATP increase 10-fold, while Km for HCO3- is increased 100-fold. Significantly, NH3 decreases rather than stimulates Pi release from ATP in the HCO3(-)-dependent ATPase reaction. The increase in kcat of the HCO3(-)-dependent ATPase reaction, and an impaired ability of the Lys841 enzyme to catalyze the reaction of NH3 with carboxy phosphate, strongly argues for interactions between the two catalytic ATP sites that couple the formation of enzyme-bound carbamate with its phosphorylation.     

Processing of proteins by the molecular chaperone Hsp104

The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.     

Structure of a genetically engineered molecular motor

Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. We present the 2.8 A resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 130 A conformational amplifier consisting of repeats 1 and 2 of alpha-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region. Disruption of this salt bridge, which is known to severely inhibit ATP hydrolysis activity, appears to interfere with formation of myosin's catalytically active 'closed' conformation. Finally, we describe the structure of alpha-actinin repeats 1 and 2 as being composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. This fold is very similar to the previously described structures of alpha-actinin repeats 2 and 3, and alpha-spectrin repeats 16 and 17.     

A flipped ion pair at the dynein–microtubule interface is critical for dynein motility and ATPase activation

Dynein is a motor protein that moves on microtubules (MTs) using the energy of adenosine triphosphate (ATP) hydrolysis. To understand its motility mechanism, it is crucial to know how the signal of MT binding is transmitted to the ATPase domain to enhance ATP hydrolysis. However, the molecular basis of signal transmission at the dynein–MT interface remains unclear. Scanning mutagenesis of tubulin identified two residues in α-tubulin, R403 and E416, that are critical for ATPase activation and directional movement of dynein. Electron cryomicroscopy and biochemical analyses revealed that these residues form salt bridges with the residues in the dynein MT-binding domain (MTBD) that work in concert to induce registry change in the stalk coiled coil and activate the ATPase. The R403-E3390 salt bridge functions as a switch for this mechanism because of its reversed charge relative to other residues at the interface. This study unveils the structural basis for coupling between MT binding and ATPase activation and implicates the MTBD in the control of directional movement.     

Importance of two ATP-binding sites for oligomerization, ATPase activity and chaperone function of mitochondrial Hsp78 protein.

The yeast mitochondrial chaperone Hsp78, a homologue of yeast cytosolic Hsp104 and bacterial ClpB, is required for maintenance of mitochondrial functions under heat stress. Here, Hsp78 was purified to homogeneity and shown to form a homo-hexameric complex, with an apparent molecular mass of approximately 440 kDa, in an ATP-dependent manner. Analysis of its ATPase activity reveals that the observed positive cooperativity effect depends both on Hsp78 and ATP concentration. Site-directed mutagenesis of the two putative Hsp78 nucleotide-binding domains suggest that the first nucleotide-binding domain is responsible for ATP hydrolysis and the second one for protein oligomerization. Studies on the chaperone activity of Hsp78 show that its cooperation with the mitochondrial Hsp70 system, consisting of Ssc1p, Mdj1p and Mge1p, is needed for the efficient reactivation of substrate proteins. These studies also suggest that the oligomerization but not the Hsp78 ATPase activity is essential for its chaperone activity.     

The post-rigor structure of myosin VI and implications for the recovery stroke

Myosin VI has an unexpectedly large swing of its lever arm (powerstroke) that optimizes its unique reverse direction movement. The basis for this is an unprecedented rearrangement of the subdomain to which the lever arm is attached, referred to as the converter. It is unclear at what point(s) in the myosin VI ATPase cycle rearrangements in the converter occur, and how this would effect lever arm position. We solved the structure of myosin VI with an ATP analogue (ADP.BeF3) bound in its nucleotide-binding pocket. The structure reveals that no rearrangement in the converter occur upon ATP binding. Based on previously solved myosin structures, our structure suggests that no reversal of the powerstroke occurs during detachment of myosin VI from actin. The structure also reveals novel features of the myosin VI motor that may be important in maintaining the converter conformation during detachment from actin, and other features that may promote rapid rearrangements in the structure following actin detachment that enable hydrolysis of ATP.     

Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein

Cytoplasmic dynein is a microtubule-based motor protein that is responsible for most intracellular retrograde transports along microtubule filaments. The motor domain of dynein contains six tandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first four containing predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiple nucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains in which mutations were introduced to block nucleotide binding at each of the four AAA modules, and then examined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state even in the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity for microtubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, but retained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-fold slower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparent binding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubule sliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essential for the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysis sites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but is critical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.     

Mutations define cross-talk between the N-terminal nucleotide-binding domain and transmembrane helix-2 of the yeast multidrug transporter Pdr5: possible conservation of a signaling interface for coupling ATP hydrolysis to drug transport

The yeast Pdr5 multidrug transporter is an important member of the ATP-binding cassette superfamily of proteins. We describe a novel mutation (S558Y) in transmembrane helix 2 of Pdr5 identified in a screen for suppressors that eliminated Pdr5-mediated cycloheximide hyper-resistance. Nucleotides as well as transport substrates bind to the mutant Pdr5 with an affinity comparable with that for wild-type Pdr5. Wild-type and mutant Pdr5s show ATPase activity with comparable K(m)((ATP)) values. Nonetheless, drug sensitivity is equivalent in the mutant pdr5 and the pdr5 deletion. Finally, the transport substrate clotrimazole, which is a noncompetitive inhibitor of Pdr5 ATPase activity, has a minimal effect on ATP hydrolysis by the S558Y mutant. These results suggest that the drug sensitivity of the mutant Pdr5 is attributable to the uncoupling of NTPase activity and transport. We screened for amino acid alterations in the nucleotide-binding domains that would reverse the phenotypic effect of the S558Y mutation. A second-site mutation, N242K, located between the Walker A and signature motifs of the N-terminal nucleotide-binding domain, restores significant function. This region of the nucleotide-binding domain interacts with the transmembrane domains via the intracellular loop-1 (which connects transmembrane helices 2 and 3) in the crystal structure of Sav1866, a bacterial ATP-binding cassette drug transporter. These structural studies are supported by biochemical and genetic evidence presented here that interactions between transmembrane helix 2 and the nucleotide-binding domain, via the intracellular loop-1, may define at least part of the translocation pathway for coupling ATP hydrolysis to drug transport.     

The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA

ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.     

Molecular and genetic analysis of condensin function in vertebrate cells

We engineered mutants into residues of SMC2 to dissect the role of ATPase function in the condensin complex. These residues are predicted to be involved in ATP binding or hydrolysis and in the Q-loop, which is thought to act as a mediator of conformational changes induced by substrate binding. All the engineered ATPase mutations resulted in lethality when introduced into SMC2 null cells. We found that ATP binding, but not hydrolysis, is essential to allow stable condensin association with chromosomes. How SMC proteins bind and interact with DNA is still a major question. Cohesin may form a ring structure that topologically encircles DNA. We examined whether condensin behaves in an analogous way to its cohesin counterpart, and we have generated a cleavable form of biologically active condensin with PreScission protease sites engineered into the SMC2 protein. This has allowed us to demonstrate that topological integrity of the SMC2-SMC4 heterodimer is not necessary for the stability of the condensin complex in vitro or for its stable association with mitotic chromosomes. Thus, despite their similar molecular organization, condensin and cohesin exhibit fundamental differences in their structure and function.     

Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs that brings the N- and C-domains of PGK closer together. Here we present the 1.8A crystal structure of unliganded PGK from Thermus caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation.     

Molecular mechanism of bacterial Hsp90 pH‐dependent ATPase activity

Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated open‐to‐closed‐to‐open conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pH‐dependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformation‐specific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anti‐correlation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255‐ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255‐ATP salt‐bridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pH‐dependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal site‐specific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity.     

Theoretical studies of the ATP hydrolysis mechanism of myosin

The ATP hydrolysis mechanism of myosin was studied using quantum chemical (QM) and molecular dynamics calculations. The initial model compound for QM calculations was constructed on the basis of the energy-minimized structure of the myosin(S1dc)-ATP complex, which was determined by molecular mechanics calculations. The result of QM calculations suggested that the ATP hydrolysis mechanism of myosin consists of a single elementary reaction in which a water molecule nucleophilically attacked gamma-phosphorus of ATP. In addition, we performed molecular dynamics simulations of the initial and final states of the ATP hydrolysis reaction, that is, the myosin-ATP and myosin-ADP.Pi complexes. These calculations revealed roles of several amino acid residues (Lys185, Thr186, Ser237, Arg238, and Glu459) in the ATPase pocket. Lys185 maintains the conformation of beta- and gamma-phosphate groups of ATP by forming the hydrogen bonds. Thr186 and Ser237 are coordinated to a Mg(2+) ion, which interacts with the phosphates of ATP and therefore contributes to the stabilization of the ATP structure. Arg238 and Glu459, which consisted of the gate of the ATPase pocket, retain the water molecule acting on the hydrolysis at the appropriate position for initiating the hydrolysis.     

Mechanism of nucleotide sensing in group II chaperonins

Group II chaperonins mediate protein folding in an ATP-dependent manner in eukaryotes and archaea. The binding of ATP and subsequent hydrolysis promotes the closure of the multi-subunit rings where protein folding occurs. The mechanism by which local changes in the nucleotide-binding site are communicated between individual subunits is unknown. The crystal structure of the archaeal chaperonin from Methanococcus maripaludis in several nucleotides bound states reveals the local conformational changes associated with ATP hydrolysis. Residue Lys-161, which is extremely conserved among group II chaperonins, forms interactions with the γ-phosphate of ATP but shows a different orientation in the presence of ADP. The loss of the ATP γ-phosphate interaction with Lys-161 in the ADP state promotes a significant rearrangement of a loop consisting of residues 160-169. We propose that Lys-161 functions as an ATP sensor and that 160-169 constitutes a nucleotide-sensing loop (NSL) that monitors the presence of the γ-phosphate. Functional analysis using NSL mutants shows a significant decrease in ATPase activity, suggesting that the NSL is involved in timing of the protein folding cycle.     

Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase

Substitution of Escherichia coli F(1)F(0) ATP synthase residues betaD372 or gammaS12 with groups that are unable to form a hydrogen bond at this location decreased ATP synthase-dependent cell growth by 2 orders of magnitude, eliminated the ability of F(1)F(0) to catalyze ATPase-dependent proton pumping in inverted E. coli membranes, caused a 15-20% decrease in the coupling efficiency of the membranes as measured by the extent of succinate-dependent acridine orange fluorescence quenching, but increased soluble F(1)-ATPase activity by about 10%. Substitution of gammaK9 to eliminate the ability to form a salt bridge with betaD372 decreased soluble F(1)-ATPase activity and ATPase-driven proton pumping by 2-fold but had no effect on the proton gradient induced by addition of succinate. Mutations to eliminate the potential to form intersubunit hydrogen bonds and salt bridges between other less highly conserved residues on the gamma subunit N-terminus and the beta subunits had little effect on ATPase or ATP synthase activities. These results suggest that the betaD372-gammaK9 salt bridge contributes significantly to the rate-limiting step in ATP hydrolysis of soluble F(1) while the betaD372-gammaS12 hydrogen bond may serve as a component of an escapement mechanism for ATP synthesis in which alphabetagamma intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven gamma subunit rotation.     

Kinetic effects of kinesin switch I and switch II mutations

We have examined several mutants in the switch I, switch II region of rat kinesin. Pre-steady-state kinetic analysis of association and dissociation of an N256K mutant with nucleotides and microtubules demonstrates that the mutation blocks microtubule stimulation of nucleotide release and ATP hydrolysis without affecting other kinetic parameters. The results suggest that ADP release on one head may be coupled to structural changes on the other head to stimulate ATP hydrolysis. Mutations at Glu(237), a residue predicted to participate in a hydrogen-bond interaction critical for nucleotide processing, reduced or abolished microtubule-dependent ATPase activity with only minor effects on pre-steady-state rates of nucleotide release or binding. Mutations at Glu(200), a residue that could serve as an alternate electron acceptor in the above-mentioned hydrogen-bond interaction, had small effects on microtubule-dependent ATPase activity despite modestly reducing the rate at which microtubule-stimulated nucleotide release occurs. These results further clarify the pathway of coupling of ATP hydrolysis to force production.