How does kinesin-1 move down a microtubule? Optical trap and fluorescence-labeling techniques similar to those used to characterize myosin (see Figures 17-28 and 17-29) have been used to study how kinesin-1 moves down a microtubule and how ATP hydrolysis is converted into mechanical work. Such experiments demonstrate that it is a very processive motor, taking hundreds of “steps” walking “hand over hand” down a microtubule without dissociating from it. During this process, the double-headed molecule takes 8-nm steps from one tubulin dimer to the next, tracking down the same protofilament within the microtubule. This movement entails each individual head taking 16-nm steps. The two heads work in a highly coordinated manner so that one is always attached to the microtubule.

The ATP cycle of kinesin-1 movement is most easily understood if we begin just after the motor has taken a step (Figure 18-21a). At this point, the motor has a nucleotide-free leading head, which is strongly bound to a tubulin dimer in a protofilament, and an ADP-bound trailing head, which is weakly associated with the protofilament. ATP then binds to the leading head (Figure 18-21a, step 1), and this binding induces a conformational change in the linker domain so that instead of pointing backward, it swings forward and “docks” into its associated head. This swinging motion results in the linker domain rotating forward, and because it is attached to the trailing head, it swings the trailing head—like throwing a ballet dancer—into position to become the leading head (Figure 18-21a, step 2). The new leading head finds the next binding site on the microtubule (Figure 18-21a, step 3 and Figure 18-21b). The binding of the leading head to the microtubule induces the leading head to release ADP while the trailing head hydrolyzes ATP to ADP and P_i, releasing P_i (Figure 18-21a, step 4). ATP can now bind to the leading head to repeat the cycle and allow the protein to take another step down the microtubule.

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Two features of this cycle ensure that one head is always firmly bound to the microtubule. First, the head domain binds tightly to the microtubule in the nucleotide-free, ATP, and ADP + P_i states, but weakly in the ADP state. Second, the two heads communicate: when the leading head binds the microtubule and releases ADP, it is converted from a weak to a tight binding state. This change is communicated to the ATP-bound trailing head, which is tightly associated with the microtubule. The trailing head is stimulated to hydrolyze ATP, releasing P_i and converting to a weak binding state. Because this cycle requires that one head always be firmly attached to a tubulin dimer in a protofilament, kinesin-1 can take thousands of steps along a microtubule without disassociating from it.

As a transporter of organelles, kinesin-1 must bind and transport the correct cargo. It does this through receptor proteins on the appropriate organelle that bind to the tail domain of the motor protein. Since kinesin-1 is an ATPase, it is important that this activity be inactivated to conserve energy when it is not needed, and also after releasing the organelle following transport. To accomplish this, kinesin-1 can fold into an inactive state in which the tail domain interacts with the motor head domain to inhibit both its ATPase activity and microtubule binding (Figure 18-22). When the tail binds the cargo-associated receptor protein, the motor unfolds and is activated (step 1). When the cargo-activated motor complex encounters a microtubule, it transports the organelle (step 2). Upon organelle delivery, kinesin-1 is released from the cargo and is inactivated by refolding into the head-to-tail inhibited state.

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When the x-ray crystallographic structure of the kinesin head was determined, it revealed a major surprise—the catalytic core has the same overall structure as myosin’s (Figure 18-23)! This is the case despite the fact that there is no amino acid sequence conservation between the two proteins, arguing strongly that convergent evolution has twice generated a fold that can use the hydrolysis of ATP to generate work. Moreover, the same type of three-dimensional structure is seen in small GTP-binding proteins, such as Ras, that undergo a conformational change upon GTP hydrolysis (see Figure 15-7).