We have identified dimeric kinesin mutants that become stalled on the microtubule after one ATP turnover, unable to bind and hydrolyze ATP at their second site. We have used these mutants to determine the regulatory signal that allows ATP to bind to the forward head, such that processive movement can continue. The results show that phosphate release occurs from the rearward head before detachment, and detachment triggers active-site accessibility for ATP binding at the forward head. This mechanism, in which the rearward head controls the behavior of the forward head, may be conserved among processive motors.
Conventional kinesin is a highly processive, plus-enddirected microtubule-based motor that drives membranous organelles toward the synapse in neurons. Although recent structural, biochemical, and mechanical measurements are beginning to converge into a common view of how kinesin converts the energy from ATP turnover into motion, it remains difficult to dissect experimentally the intermolecular domain cooperativity required for kinesin processivity. We report here our presteady-state kinetic analysis of a kinesin switch I mutant at Arg 210 (NXXSSRSH, residues 205-212 in Drosophila kinesin). The results show that the R210A substitution results in a dimeric kinesin that is defective for ATP hydrolysis and a motor that cannot detach from the microtubule although ATP binding and microtubule association occur. We propose a mechanistic model in which ATP binding at head 1 leads to the plus-end-directed motion of the neck linker to position head 2 forward at the next microtubule binding site. However, ATP hydrolysis is required at head 1 to lock head 2 onto the microtubule in a tight binding state before head 1 dissociation from the microtubule. This mechanism optimizes forward movement and processivity by ensuring that one motor domain is tightly bound to the microtubule before the second can detach.Kinesin is a highly processive, dimeric mechanoenzyme that travels along microtubules toward their plus-ends in discrete 8-nm steps, each step tightly coupled to a single ATP turnover (1-3). Recent evidence from a variety of experimental approaches has focused our attention to the proposal presented by Rice et al. (4) that ATP binding induces a pronounced conformational change in the neck linker region, which docks the neck linker onto the catalytic core and propels the unattached kinesin head forward to find the next binding site on the microtubule. This model is based on a disorder-to-order transition in the neck linker region for monomeric kinesin constructs. The neck linker of the Mt⅐K 1 complex was shown to be mobile in the presence of ADP, existing in an equilibrium with two predominant conformations trapped by cryo-electron microscopy. However, upon the addition of ATP or nonhydrolyzable ATP analogs to the Mt⅐K complex, the neck mobility ceased with the neck linker element tightly associated with the catalytic core. This ordered state was reversed by the addition of ADP or loss of nucleotide. In addition, the cryo-electron microscopy of this proposed ATP state revealed a single discrete orientation of the neck linker with the carboxyl terminus of the motor domain directed toward the plus-end of the microtubule (4). Xing et al. (5) have reported for a monomeric kinesin motor domain two discrete structural transitions induced by ADP binding and another produced by ATP binding. These three conformations revealed by fluorescence resonance energy transfer were consistent with the results reported by Rice et al. (4). Furthermore, biochemical studies of dimeric kinesin have demonstrated that ATP binding (or nonhydrolyzabl...
Conventional kinesin is a highly processive, microtubule-based motor protein that drives the movement of membranous organelles in neurons. Using in vivo genetics in Drosophila melanogaster, Glu 164 was identified as an amino acid critical for kinesin function [Brendza, K. M., Rose, D. ]. Glu 164 is located at the -strand 5a/loop 8b junction of the catalytic core and projects toward the microtubule binding face in close proximity to key residues on -tubulin helix R12. Substitution of Glu 164 with alanine (E164A) results in a dimeric kinesin with a dramatic reduction in the microtubule-activated steady-state ATPase (5 s -1 per site versus 22 s -1 per site for wild-type). Our analysis shows that E164A binds ATP and microtubules with a higher affinity than wild-type kinesin. The rapid quench and stopped-flow results provide evidence that ATP hydrolysis is significantly faster and the precise coordination between the motor domains is disrupted. The data reveal an E164A intermediate that is stalled on the microtubule and cannot bind and hydrolyze ATP at the second head.Conventional kinesin is a processive, dimeric microtubule motor protein that travels toward the plus end of the microtubule in 8 nm steps (reviewed in refs 1-5). The processive steps require coordination between the two motor domains and with the microtubule, which is achieved through the ATPase cycle (6-13). The amino acid sequence of the nucleotide binding motifs and the crystal structure of the catalytic core show homology with other kinesin superfamily members, myosins, and G proteins (P-loop nucleotidases). These results suggest a common mechanism through switch I and switch II that translates the state of the nucleotide bound at the catalytic core into structural transitions to communicate with protein partners (14-23; reviewed in refs 24-27).C-Terminal to the catalytic core of kinesin is the neck region, which consists of two short strands (neck linker) followed by a coiled-coil helix (neck coiled-coil) (16). The neck linker controls the plus-end directionality of kinesin movement (20,(28)(29)(30)(31). In addition, crystal structures of dimeric kinesin and Ncd docked on the microtubule by cryo-EM 1 reconstruction (16,20,(32)(33)(34)(35)(36)(37) show specific neck linker conformations for the plus-end and minus-end directed dimeric motors and position them differently on the microtubule. The kinesin neck linker interacts with the catalytic core near microtubule binding loop L12 and the switch II relay helix, R4, which is involved in the communication between the nucleotide and microtubule binding sites (31, 38). Rice et al. (31) showed for a kinesin monomer that ATP promoted neck linker docking onto the catalytic core and the orientation was toward the plus end of the microtubule. ATP hydrolysis resulted in the neck linker returning to a more mobile conformation. On the basis of these results, Rice et al. proposed that the energy from ATP binding is coupled to neck linker docking onto the catalytic core. They suggested that the structural transiti...
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