Stepping and Stretching

Abstract

The ability of kinesin to travel long distances on its microtubule track without dissociating has led to a variety of models to explain how this remarkable degree of processivity is maintained. All of these require that the two motor domains remain enzymatically "out of phase," a behavior that would ensure that, at any given time, one motor is strongly attached to the microtubule. The maintenance of this coordination over many mech-anochemical cycles has never been explained, because key steps in the cycle could not be directly observed. We have addressed this issue by applying several novel spectroscopic approaches to monitor motor dissocia-tion, phosphate release, and nucleotide binding during processive movement by a dimeric kinesin construct. Our data argue that the major effect of the internal strain generated when both motor domains of kinesin bind the microtubule is to block ATP from binding to the leading motor. This effect guarantees the two motor domains remain out of phase for many mechanochemi-cal cycles and provides an efficient and adaptable mechanism for the maintenance of processive movement. Members of the kinesin family of molecular motors are capable of taking over 100 steps on their microtubule track without dissociating, a feature that would be necessary for a transport motor that operates in isolation (1-6). A variety of kinetic, structural, and mechanical studies have revealed that this processive behavior requires that two motors of kinesin remain in different structural and enzymatic states during a proces-sive run (7-10, 11, 12). This would ensure that, at any given time, at least one of the two heads would remain strongly attached to its track, preventing the motor from prematurely detaching. Such coordination requires a way for the two motor domains to communicate their structural states to each other while walking processively. Several lines of evidence suggest that this allosteric communication is mediated through the internal load generated when both heads attach to the micro-tubule (1, 13-16). As illustrated below in Fig. 1, kinesin initiates its mechanochemical cycle with its attached head (green) nucleotide free and its tethered head (magenta) containing ADP in the active site. ATP binding to the attached head reorients its neck linker (blue), which swings the tethered head forward to the next tubulin-docking site. ADP is then released from the new, weakly bound leading head (magenta) to produce an intermediate in which both heads are strongly bound to the microtubule. This situation would generate rearward strain on the neck linker of the leading head, depicted as a left pointing arrow, and forward strain on the corresponding structure of the trailing head, depicted as a right pointing arrow. It has been proposed that this strain generates processivity by accelerating release of the trailing head (13, 17). In this mechanism, release of the ADP-containing trailing head would be very slow in the absence of forward strain and fast in its presence. In such a system, the greater that forward strain accelerates k dMT , the greater the degree of processivity. However , there is an internal inconsistency with this scheme. If kinesin's processivity were dependent solely on this mechanism , the motor would dissociate from the microtubule after only a few steps. The reasoning behind this is illustrated in Fig. 1. We have recently shown (18) that the effective rate of trailing head dissociation (k dMT 50 s 1) is appreciably slower than that for ADP release (k dADP 170 s 1 , this work, and Refs. 8, 19-21), and ATP hydrolysis (k h , 100 s 1 , Refs. 8, 19, 21, 22). This would lead to accumulation of a kinesin intermediate with both heads attached to the microtubule, with the leading head nucleotide free, and with ADP-P i in the active site of the trailing head. Given millimolar intracellular ATP concentrations and an apparent second order rate constant of 1 M 1 s 1 (2, 8, 15, 19, 20, 23), ATP would then rapidly bind to the new leading head (1000 s 1) and be hydrolyzed. This would generate an intermediate with both heads weakly bound, and dissociation would rapidly follow, as indicated in Fig. 1 by the red X. The fact that kinesin is highly processive (1-6) argues that there is a mechanism that prevents it from proceeding down this pathway. An alternative possibility is that rearward strain on the leading head slows ATP binding and subsequent hydrolysis, insuring that the leading head would remain strongly attached until the trailing head had dissociated. ATP would then rapidly bind to the leading head and cause the trailing head to swing forward to the next tubulin-docking site. Processive movement would be favored, because the rate of this forward stepping movement, at 800 s 1 , is nearly sixteen times faster than the rate of trailing head dissociation (18). Furthermore, blocking ATP binding to the forward head while it was experiencing rearward strain would prevent the motor from proceeding down the pathway marked by the red X in Fig. 1. Determining whether processivity depends on the first mechanism , the second, or to some degree on both requires the ability to unambiguously measure the rates of key steps in the mechanochemical cycle and the effects of strain on these rates. These include the rates of trailing head dissociation, of ADP