High-performance fungal motors.
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Adenosine Triphosphate↗
Biomedical subjects
S P Gilbert
Publications and source records attributed to S P Gilbert.
A mechanistic model for Ncd directionality.
Ncd is a kinesin-related protein that drives movement to the minus-end of microtubules. Pre-steady-state kinetic experiments have been employed to investigate the cooperative interactions between the motor domains of the MC1 dimer and to establish the ATPase mechanism. Our results indicate that the active sites of dimeric Ncd free in solution are not equivalent; ADP is held more tightly at one site than at the other. Upon microtubule binding, fast release of ADP from the first motor domain is stimulated at 18 s(-1), yet rate-limiting ADP release from the second motor domain occurs at 1.4 s(-1). We propose that the head with the low affinity for ADP binds the microtubule first to establish the directional bias of the microtubule.Ncd intermediate where one motor domain is bound to the microtubule with the second head detached and directed toward the minus-end of the microtubule. The force generating cycle is initiated as ATP binds to the empty site of the microtubule-bound head. ATP hydrolysis at head 1 is required for head 2 to bind to the microtubule. The kinetics indicate that two ATP molecules are required for a single step and force generation for minus-end directed movement generated by this non-processive dimeric motor.
Adenosine Diphosphate↗
ATP-dependent simian virus 40 T-antigen-Hsc70 complex formation.
Simian virus 40 large T antigen is a multifunctional oncoprotein that is required for numerous viral functions and the induction of cellular transformation. T antigen contains a J domain that is required for many of its activities including viral DNA replication, transformation, and virion assembly. J-domain-containing proteins interact with Hsc70 (a cellular chaperone) to perform multiple biological activities, usually involving a change in the conformation of target substrates. It is thought that Hsc70 associates with T antigen to assist in performing its numerous activities. However, it is not clear if T antigen binds to Hsc70 directly or induces the binding of Hsc70 to other T-antigen binding proteins such as pRb or p53. In this report, we show that T antigen binds Hsc70 directly with a stoichiometry of 1:1 (dissociation constant = 310 nM Hsc70). Furthermore, the T-antigen--Hsc70 complex formation is dependent upon ATP hydrolysis at the active site of Hsc70 (ATP dissociation constant = 0.16 microM), but T-antigen--Hsc70 complex formation does not require nucleotide hydrolysis at the T-antigen ATP binding site. N136, a J domain-containing fragment of T antigen, does not stably associate with Hsc70 but can form a transient complex as assayed by centrifugation analysis. Finally, T antigen does not associate stably with either of two yeast Hsc70 homologues or an amino-terminal fragment of Hsc70 containing the ATPase domain. These results provide direct evidence that the T-antigen--Hsc70 interaction is specific and that this association requires multiple domains of both T antigen and Hsc70. This is the first demonstration of a nucleotide requirement for the association of T antigen and Hsc70 and lays the foundation for future reconstitution studies of chaperone-dependent tumorigenesis induced by T antigen.
Adenosine Triphosphate↗
A kinesin mutation that uncouples motor domains and desensitizes the gamma-phosphate sensor.
Conventional kinesin is a processive, microtubule-based motor protein that drives movements of membranous organelles in neurons. Amino acid Thr(291) of Drosophila kinesin heavy chain is identical in all superfamily members and is located in alpha-helix 5 on the microtubule-binding surface of the catalytic motor domain. Substitution of methionine at Thr(291) results in complete loss of function in vivo. In vitro, the T291M mutation disrupts the ATPase cross-bridge cycle of a kinesin motor/neck construct, K401-4 (Brendza, K. M., Rose, D. J., Gilbert, S. P., and Saxton, W. M. (1999) J. Biol. Chem. 274, 31506-31514). The pre-steady-state kinetic analysis presented here shows that ATP binding is weakened significantly, and the rate of ATP hydrolysis is increased. The mutant motor also fails to distinguish ATP from ADP, suggesting that the contacts important for sensing the gamma-phosphate have been altered. The results indicate that there is a signaling defect between the motor domains of the T291M dimer. The ATPase cycles of the two motor domains appear to become kinetically uncoupled, causing them to work more independently rather than in the strict, coordinated fashion that is typical of kinesin.
Adenosine Triphosphatases↗
Kinetic studies of dimeric Ncd: evidence that Ncd is not processive.
Ncd is a kinesin-related motor protein which drives movement to the minus-end of microtubules. The kinetics of Ncd were investigated using the dimeric construct MC1 (Leu(209)-Lys(700)) expressed in Escherichia coli strain BL21(DE) as a nonfusion protein [Chandra, R., Salmon, E. D., Erickson, H. P., Lockhart, A., and Endow, S. A. (1993) J. Biol. Chem. 268, 9005-9013]. Acid chemical quench flow methods were used to measure directly the rate of ATP hydrolysis, and stopped-flow kinetic methods were used to determine the kinetics of mantATP binding, mantADP release, dissociation of MC1 from the microtubule, and binding of MC1 to the microtubule. The results define a minimal kinetic mechanism, M.N + ATP M.N.ATP M.N.ADP.P N. ADP.P N.ADP + P M.N.ADP M.N + ADP, where N, M, and P represent Ncd, microtubules, and inorganic phosphate respectively, with k(+1) = 2.3 microM(-1) s(-1), k(+2) =23 s(-1), k(+3) =13 s(-1), k(+5)= 0.7 microM(-)(1) s(-)(1), and k(+6) = 3.7 s(-)(1). Phosphate release (k(+4)) was not measured directly although it is assumed to be fast relative to ADP release because Ncd is purified with ADP tightly bound at the active site. ATP hydrolysis occurs at 23 s(-)(1) prior to Ncd dissociation at 13 s(-)(1). The pathway for ATP-promoted detachment (steps 1-3) of Ncd from the microtubule is comparable to kinesin's. However, there are two major differences between the mechanisms of Ncd and kinesin. In contrast to kinesin, mantADP release for Ncd at 3.7 s(-)(1) is the slowest step in the pathway and is believed to limit steady-state turnover. Additionally, the burst amplitude observed in the pre-steady-state acid quench experiments is stoichiometric, indicating that Ncd, in contrast to kinesin, is not processive for ATP hydrolysis.
Adenosine Triphosphatases↗
Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd.
Ncd is a minus-end-directed microtubule motor and a member of the kinesin superfamily. The Ncd dimer contains two motor domains, and cooperative interactions between the heads influence the interactions of each respective motor domain with the microtubule. The approach we have taken to understand the cooperativity between the two motor domains is to analyze the ATPase cycle of dimeric MC1 and monomeric MC6. The steps in the ATPase cycle where cooperativity occurs can be identified by comparing the two mechanisms. The rate-limiting step in the MC6 mechanism is ADP release at 3.4 s(-)(1). The observed rate constant for ATP-induced dissociation from the microtubule is 14 s(-)(1). However, the relative amplitude associated with MC6 dissociation is extremely small in comparison to the amplitude associated with dimeric MC1 dissociation kinetics. The amplitude data indicate that monomeric MC6 does not detach from the microtubule during the initial turnovers of ATP, and ATP hydrolysis is uncoupled from movement. The results show that cooperative interactions between the motor domains of the dimer are required for ATP-dependent dissociation; therefore, one function of the partner motor domain may be to weaken the interaction of the adjacent head with the microtubule.
Adenosine Diphosphate↗
Kinetics: a tool to study molecular motors.
Molecular motors are enzymes that couple the energy from nucleoside triphosphate hydrolysis to movement along a filament lattice. The three cytoskeletal motor superfamilies include myosin, dynein, and kinesin. However, in the last decade it has become apparent that the nucleic acid-based enzymes (DNA and RNA polymerases as well as the DNA helicases) share a number of mechanistic features in common with the microtubule and actin motors despite the fact that their cellular functions are so different. This review addresses the mechanistic approaches that have been used to study molecular motors. We discuss the basic biochemical techniques used to characterize a protein preparation, including active site determination and steady-state kinetics. In addition, we present the transient-state kinetic approaches used to define a mechanochemical cycle. We attempt to integrate the information obtained from kinetic studies within the context of motility results to provide a better understanding of the contribution of each approach for dissecting unidirectional force generation.
Adenosine Triphosphate↗
Lethal kinesin mutations reveal amino acids important for ATPase activation and structural coupling.
To study the relationship between conventional kinesin's structure and function, we identified 13 lethal mutations in the Drosophila kinesin heavy chain motor domain and tested a subset for effects on mechanochemistry. S246F is a moderate mutation that occurs in loop 11 between the ATP- and microtubule-binding sites. While ATP and microtubule binding appear normal, there is a 3-fold decrease in the rate of ATP turnover. This is consistent with the hypothesis that loop 11 provides a structural link that is important for the activation of ATP turnover by microtubule binding. T291M is a severe mutation that occurs in alpha-helix 5 near the center of the microtubule-binding surface. It impairs the microtubule-kinesin interaction and directly effects the ATP-binding pocket, allowing an increase in ATP turnover in the absence of microtubules. The T291M mutation may mimic the structure of a microtubule-bound, partially activated state. E164K is a moderate mutation that occurs at the beta-sheet 5a/loop 8b junction, remote from the ATP pocket. Surprisingly, it causes both tighter ATP-binding and a 2-fold decrease in ATP turnover. We propose that E164 forms an ionic bridge with alpha-helix 5 and speculate that it helps coordinate the alternating site catalysis of dimerized kinesin heavy chain motor domains.
Adenosine Triphosphatases↗
The diversity of molecular motors: an overview.
Rapid progress has recently been made in the identification and characterization of a large number of kinesin and myosin motor proteins. Recent work has uncovered roles for these motors in processes such as vesicle trafficking, cytoskeletal organization, and chromosome movements, to name a few. A series of reviews describing some of the significant advances in our understanding of the structure and function of myosins and kinesins is presented.
Actins↗
Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein.
Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.
Adenosine Triphosphate↗
Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains.
Non-claret disjunctional protein (Ncd) is a minus end-directed microtubule motor required for normal spindle assembly and integrity during Drosophila oogenesis. We have pursued equilibrium binding experiments to examine the affinity of Ncd for microtubules in the presence of the ATP nonhydrolyzable analog 5'-adenylyl-beta, gamma-imidodiphosphate (AMP-PNP), ADP, or ADP + Pi using both dimeric (MC1) and monomeric (MC6) Ncd constructs expressed in Escherichia coli. Both MC1 and MC6 sediment with microtubules in the absence of added nucleotide as well as in the presence of either ADP or AMP-PNP. Yet, in the presence of ADP + Pi, there is a decrease in the affinity of both MC1 and MC6 for microtubules. The data for dimeric MC1 show that release of the dimer to the supernatant is sigmoidal with the apparent Kd(Pi) for the two phosphate sites at 23.3 and 1.9 mM, respectively. The results indicate that binding at the first phosphate site enhances binding at the second site, thus cooperatively stimulating release. Stopped-flow kinetics indicate that MgATP promotes dissociation of the Mt.MC1 complex at 14 s-1, yet AMP-PNP has no effect on the Mt.MC1 complex. These results are consistent with a model for the ATPase cycle in which ATP hydrolysis occurs on the microtubule followed by detachment as the Ncd.ADP.Pi intermediate.
Adenosine Diphosphate↗
Alternating site mechanism of the kinesin ATPase.
The processivity of the microtubule-kinesin ATPase has been investigated using stopped-flow kinetic methods to measure the binding of each motor domain of the dimeric kinesin (K401) to the microtubule and the release of the fluorescent ADP analog, 2'(3')-O-(N-methylanthraniloyl)adenosine 5'-diphosphate (mantADP) from the active site of the motor domain. The results show that the release of two molecules of ADP from dimeric kinesin (K401) after the binding of kinesin ADP to the microtubule is a sequential process leading to biphasic kinetics. The maximum rate of release of mantADP from the first motor domain of K401 or monomeric K341 is fast (300 s-1) and independent of added nucleotide. The rate of mantADP release from the second motor domain of K401 is slow in the absence of added nucleotide (0.4 s-1) and reaches a maximum rate of 300 s-1 at saturating concentrations of ATP. High concentrations of ADP stimulate mantADP release from the second head to a maximum rate of 3.8 s-1. The nonhydrolyzable analog AMP-PNP and ATP-gamma S also stimulate ADP release from the second head (maximum rate of 30 s-1), suggesting that ATP hydrolysis is not necessary to stimulate the ADP release. These experiments establish an alternating site mechanism for dimeric kinesin whereby ATP binding to one kinesin active site stimulates the release of ADP from the second site such that the reactions occurring at the active sites of the two monomer units are kept out of phase from each other by interactions between the heads. These results define the steps of the ATPase pathway that lead to the efficient coupling of ATP hydrolysis to force production in a processive reaction whereby force production in forming a tight microtubule complex by one head is coupled to the rate-limiting release of the other head from the microtubule.
Adenosine Diphosphate↗
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.
Adenosine Diphosphate↗
Purification and characterization of two monomeric kinesin constructs.
Steady-state and pre-steady-state kinetic methods were used to analyze two shorter Drosophila kinesin constructs (K341 and K366) in comparison to K401. K341, K366, and K401 represent the kinesin motor domains containing the N-terminal 341, 366, or 401 amino acids, respectively. K401 is dimeric (Kd = 37 +/- 17 nM) whereas both K366 and K341 are monomeric [Correia et al. (1995) Biochemistry 34, 4898-4907]. Like native kinesin and K401, K341 and K366 demonstrate low ATPase activity in the absence of microtubules (0.03 and 0.01 s-1, respectively), and ADP release is rate-limiting during steady-state turnover. Microtubules activate the steady-state ATPase to 84 s-1 for K341 (K(m),ATP = 100 microM; K0.5,MT = 3.2 microM tubulin) and 64 s-1 for K366 (K(m),ATP = 65 microM; K0.5,MT = 2.5 microM tubulin) in comparison to K401 at 20 s-1 (K(m)ATP = 60 microM; K0.5,MT = 1 microM tubulin). The rapid quench experiments for all three constructs show a burst of product formation during the first turnover, indicating the rate-limiting step for the microtubule-activated ATPase occurs after ATP hydrolysis. The interaction of K341 and K366 with the microtubule was analyzed by electron microscopy. The results show that K341 and K366, like K401, bind to the microtubule with an 8 nm axial periodicity. However, the addition of K366 to microtubules resulted in significant aggregation of microtubules. The pre-steady-state kinetic results show that K341 retains the kinetic and structural properties necessary to compare directly the kinetic properties of monomeric and dimeric kinesins, although the microtubule-activated ATPase is significantly faster for the monomeric constructs, suggesting possible interactions in the dimer which inhibit ATP turnover as part of the coupling to force production.
Adenosine Triphosphatases↗
Costs of coronary restenosis (Lovastatin Restenosis Trial).
Within the Lovastatin Restenosis Trial, restenosis has been clearly shown to increase resource utilization and costs. While it is not possible to generalize these results to other patient populations, it is clear that successful efforts to decrease restenosis will certainly improve efficacy while decreasing follow-up costs and increasing the cost-effectiveness of intervention in the coronaries.
Adult↗
Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341.
Bacterial expressed kinesin motor domains hydrolyze ATP and promote microtubule-dependent motility. It has routinely been assumed that motor domain preparations are monomeric on the basis of the presumption that dimerization is mediated by the stalk region. However, experimental verification of the oligomeric state of the kinesin construct is required to interpret the results from single-molecule motility assays as well as presteady-state kinetic experiments. We have measured directly the state of assembly of three conventional kinesin motor domain constructs-K401, K366, and K341, comprising the N-terminal 401, 366, and 341 amino acids, respectively, of the Drosophila kinesin heavy chain-by sedimentation velocity and sedimentation equilibrium methods in an analytical ultracentrifuge. K401 (MW of ADP complex, 45,532) is a predominantly a dimer with a sedimentation coefficient, s020,w, of 5.06 S, but it is able to self-associate by means of a 1-2-4 mechanism into higher oligomers. Molecular weight measurements establish the dissociation constant for dimerization at 37 +/- 17 nM in the presence of ATP. The dissociation constant in the presence of ADP is 35 +/- 26 nM and in the presence of AMPPNP is 42 +/- 28 nM. The construct K366 (MW of ADP complex, 41,404) is a monomer (measured MW, 41,768 +/- 1219) at concentrations below 4 microM K366, with a sedimentation coefficient, s020,w, of 3.25 S. At higher concentrations, there is evidence for a weak association of K366 to a 1-2-4-8 model with a slight preference for octamer formation. The smallest construct, K341 (MW of ADP complex, 38,274), is a monomer (measured MW, 38,191 +/- 734) up to at least 10 microM total K341 concentration with a sedimentation coefficient, s020,w, of 2.9 S. Thus, the dimerization domain either is between amino acid residues 367 and 401 or is strongly affected by the removal of this region. Higher oligomers of K401 form by a mechanism involving dimers of dimers, and suggest that native kinesin may also undergo self-association. These results have important implications for the interpretation of ATP-dependent motility assays.
Adenosine Diphosphate↗
Pathway of processive ATP hydrolysis by kinesin.
Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin-ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin.
Adenosine Diphosphate↗
Pathway of the microtubule-kinesin ATPase.
We have established pathway of the kinesin ATPase by direct measurement of each step in the pathway. Kinesin binds to microtubules with an 8-nm repeat and a stoichiometry of one kinesin monomer unit per tubulin dimer. Thus, the dimeric kinesin binds with both heads attached to the microtubule and on adjacent tubulin subunits. In the steady state, kinesin has a low ATPase activity that is limited by the rate of ADP release (< 0.01 s-1) in the absence of microtubules and is activated 2000-fold by the addition of microtubules to achieve a maximum rate of approximately 20 s-1. Transient-state kinetic analysis has provided direct measurement of individual steps of the reaction to define the pathway of the microtubule-kinesin ATPase. These studies establish that the rate-limiting step in the ATPase pathway is the release of the kinesin-product complex (K.ADP.P) from the microtubule following ATP hydrolysis. After phosphate release, the rebinding of kinesin-ADP to the microtubule is fast, accounting for the high activation of the ATPase at low microtubule concentration. This ATPase cycle explains the phenomenological differences between myosin and kinesin observed in motility assays. Kinesin remains associated with a microtubule through multiple rounds of hydrolysis, because it spends only a small fraction of its duty cycle in the dissociated state. The discussion of this paper will focus on the new data, their interpretation, and significance for mechanisms of force production. The ATPase coupling mechanism will be compared with dynein and myosin.
Adenosine Triphosphatases↗