We have examined the energetics of the interactions of two kinesin constructs with nucleotide and microtubules to develop a structural model of kinesin-dependent motility. Dimerization of the constructs was found to reduce the maximum rate of the microtubule-activated kinesin ATPase 5-fold. Beryllium fluoride and aluminum fluoride also reduce this rate, and they increase the affinity of kinesin for microtubules. By contrast, inorganic phosphate reduces the affinity of a dimeric kinesin construct for microtubules. These findings are consistent with a model in which the kinesin head can assume one of two conformations, "strong" or "weak" binding, determined by the nature of the nucleotide that occupies the active site. Data for dimeric kinesin are consistent with a model in which kinesin.ATP binds to the microtubule in a strong state with positive cooperativity; hydrolysis of ATP to ADP+P(i) leads to dissociation of one of the attached heads and converts the second, attached head to a weak state; and dissociation of phosphate allows the second head to reattach. These results also argue that a large free energy change is associated with formation of kinesin.ADP.P(i) and that this step is the major pathway for dissociation of kinesin from the microtubule.

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Myosin I has been implicated as the motor that drives protrusion of the leading edge of motile cells. This function requires a close association with the plasma membrane and the cytoskeleton. Association with the actin cytoskeleton is mediated by an ATP-dependent binding site in the motor-containing myosin head, as well as by a second, ATP-independent actin binding site. In myosin IC from Acanthamoeba, the ATP-independent actin binding site is located in the carboxy-terminal tail, in a domain composed of two segments. The first segment is basic and is referred to as the GPA-rich segment. The second is a highly conserved sequence called src homology region 3 (SH3), found in a variety of cytoskeletal-associated proteins. We have used bacterially-expressed fusion proteins containing portions of Dictyostelium myosin IB to determine if the tail of this myosin I isoform also binds to actin and to establish precisely where the actin binding site is located. We have determined that the carboxy-terminal portion of the tail of Dictyostelium myosin IB can bind to actin in an ATP-independent manner and that the actin binding site is contained within residues 922-1059, corresponding to the GPA-rich segment of Acanthamoeba myosin IC. We conclude that this region contains a specific actin binding site which may be responsible for the cytoskeletal association of this myosin I isoform.

We have investigated the structural changes that occur in the molecular motor kinesin during its ATPase cycle, utilizing two bacterially expressed constructs. The structure of both constructs has been examined as a function of the nature of the nucleotide intermediate occupying the active site by means of sedimentation velocity, sedimentation equilibrium, fluorescence solute quenching, fluorescence anisotropy decay, and limited proteolysis. While the molecular weight of monomeric and dimeric human kinesin constructs, as measured by sedimentation velocity and sedimentation equilibrium, and the tryptic cleavage pattern are unaffected by the nucleotide intermediate occupying the active site, significant changes in the rotational correlation time of fluorescently labeled kinesin-nucleotide intermediates can be detected. These results suggest that kinesin contains an internal "hinge" whose flexibility varies through the course of the ATPase cycle. In prehydrolytic, "strong" binding states, this hinge is relatively rigid, while in posthydrolytic, "weak" binding states, it is more flexible. Our results, in conjunction with anisotropy decay studies of myosin, suggest that these two molecular motors may share a common structural feature; viz. weak binding states are characterized by segmental flexibility, which is lost upon assumption of a strong binding conformation.Kinesin is a member of a microtubule-dependent family of molecular motors that is currently a subject of intensive study in a number of laboratories (1, 2). Experiments with both native kinesin and with a variety of recombinant kinesin constructs have measured the rates and equilibrium constants for most of the elementary steps in the kinesin ATPase cycle (3-7). These studies have shown that for both the native molecule and the recombinant constructs, ADP release is rate-limiting, that microtubules accelerate both ADP and ATP release, and that kinesin dissociation from the microtubule occurs after either ATP hydrolysis or formation of a kinesin⅐ADP transition state. Compared with myosin, the rate of kinesin dissociation from the microtubule is much slower, and its rate of rebinding is much faster (6). Furthermore, kinesin is not dissociated from microtubules by the nonhydrolyzable ATP analogue AMP-PNP, 1 and in vitro motility studies have suggested that kinesin⅐ADP was a relatively "weak" binding state compared with kinesin⅐ATP (8). Thus, kinesin appears to differ from myosin in several fundamental aspects of its ATPase cycle. In vitro motility studies reveal that unlike myosin, kinesin has the capability of traveling long distances on the microtubule without dissociating (9), a feature termed "processivity." This has been attributed to the slow rate of kinesin dissociation from the microtubule and the rapid rate of its rebinding (6) or to the presence of cooperativity between the two heads of kinesin, preventing the second head of the kinesin dimer from attaching to the microtubule until the first one has detached (10).In a recent study, we have shown that ...

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