Structural Studies of Kinesin-Nucleotide Intermediates

Abstract

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 ...

Long Range Allosteric Control of Cytoplasmic Dynein ATPase Activity by the Stalk and C-terminal Domains

“…Solute Quenching and ADP Binding and Release Kinetics-The fluorescence emission of 2Ј-deoxy-mant-ADP bound to the 380-and 210-kDa fragments of dynein was measured as a function of acrylamide concentration as described previously (14). In brief, dynein fragments were mixed with a 10-fold molar excess of 2Ј-deoxy-mant-A DP, incubated overnight at 4°C, and the excess nucleotide was removed by gel filtration on pre-poured Sephadex G-25 columns (PD-10, GE Healthcare), followed by dialysis.…”

Structural and Kinetic Studies of Phosphorylation-dependent Regulation in Smooth Muscle Myosin

“…The various recombinant HMM constructs as well as protease-prepared HMM were labeled in the active site with mant ADP as described (31). Labeling of RLC with 1,5-IAEDANS was performed as described previously (31). Exchange of AEDANS-labeled RLC onto a recombinant regulatory domain construct was performed at 40°C, as described (31).…”

“…Fluorescence lifetimes and anisotropies were measured using a picosecond laser as excitation source, as described (33) in samples that had been equilibrated in 150 mM KCl, 25 mM Hepes, 1 mM MgCl 2 , 1 mM DTT, and Ϯ 0.2 mM beryllium sulfate, and 5 mM NaF. Calculation of the bound mant ADP concentration, utilizing the published extinction coefficient at 356 nm (5800 M Ϫ1 cm…”

“…The various recombinant HMM constructs as well as protease-prepared HMM were labeled in the active site with mant ADP as described (31). Labeling of RLC with 1,5-IAEDANS was performed as described previously (31).…”

“…Labeling of RLC with 1,5-IAEDANS was performed as described previously (31). Exchange of AEDANS-labeled RLC onto a recombinant regulatory domain construct was performed at 40°C, as described (31).…”