The necessity of a two-heads bound intermediate for processive movement is widely accepted, as is the fact that this requires a conformational change in the observed crystal structure [Block 1998]. Romberg et al. [1998] elaborated the theory that a reorientation of the neck-linker during the hydrolysis cycle temporarily enables double-headed binding. It involves breaking of the bonds holding the neck-linker to the catalytic core, whereupon the neck-linker adopts a flexible conformation and folds to the opposite end of the catalytic core (Figure 1.4 B). In favor of this model, Rice et al. [1999] detected an ATP-dependent conformational change of the neck-linker using electron paramagnetic resonance, fluorescence resonance energy transfer, and cryoelectron microscopy. They found that the neck-linker is docked onto the catalytic core when kinesin is bound to microtubules and ATP is in the active site, but becomes unzippered in the ADP or nucleotide-free state. These findings, together with cryoelectron microscopy work that reveals the docking orientation of kinesin on the microtubule, suggest that the two kinesin heads can span the distance between tubulin binding sites if the neck-linker of the rear head is docked and pointing forward and the neck-linker on the forward head is detached and pointing backward [Vale & Milligan 2000]. Sindelar et al. [2002] showed that the docked conformation of the neck-linker visible in some
crystal structures is comparable to the docking observed by electron paramagnetic resonance spectroscopy. The authors explained the crystallisation of docked and disordered neck-linker structures with ADP in the active site by the absence of microtubules. They concluded that only in the presence of microtubules is neck-linker flexibility dependent on the nucleotide state. The results of Rice et al. [1999] and Sindelar et al. [2002] were obtained on monomeric constructs and have been questioned to be applicable to dimers [Schief & Howard 2001]. A recent study applied cryoelectron microscopy and image reconstruction to investigate the location of the kinesin neck in dimeric and monomeric constructs complexed with microtubules [Skiniotis et al. 2003]. They confirmed the states of neck-linker mobility for monomers and showed that in dimers the neck-linker behaved identically, namely being flexible in the absence of nucleotide and ordered in the presence of AMP-PNP. Furthermore, the opposite orientation of the two neck-linkers when both heads are bound was demonstrated. Sugata et al. [2004] reached a different conclusion. The authors investigated dimeric kinesin by electron spin resonance to determine the conformation of the neck-linker in the absence and presence of microtubules. As in the studies on monomeric constructs, without microtubules the neck-linkers of dimers co-existed in both docked and disordered conformations. In all nucleotide states, however, the neck-linkers were well ordered when dimeric kinesin is bound to microtubules. These data do not support the nucleotide-dependent ‘flexibility switch’ model developed for monomeric kinesin [Rice et al. 1999] and applicable to dimeric kinesin [Skiniotis et al. 2003]. Instead a different mechanism is suggested: when both motor domains are bound to the microtubule, the neck-linkers of each motor core are fixed in different orientations. In the presence of microtubules the neck-linker does not change the degree of flexibility during the ATPase cycle, but changes its orientation between two fixed conformations. However, the authors do not comment on the difficulties to interpret the overlapping signal of two spin labels in a dimer and the slight differences in spectra observed for different nucleotides in the presence of microtubules. Whatever mechanism is correct, the key aspect of both models is the opposing orientation of the neck-linker when both motor domains are bound to the microtubule.
How do the results of the present crosslinking study fit into the current understanding of neck- linker motility?
The inability of the crosslinked neck-linker/motor core constructs to produce motility suggests that the position of the neck-linker plays a crucial role in the mechanical transducer process. Already a partial immobilisation of the neck-linker prevents movement even in the multiple motor gliding assay (4.4.2.1). This confirms the notion that it is not the coordination
of the motor domains that is impeded, but the function of the heads themselves. Monomeric kinesin constructs cannot move processively as they have only a single motor domain, but still many molecules can act in concert to move microtubules at a reduced velocity of 0.7- 1 µm/s for NcKin [Kallipolitou et al. 2001]. The minimal kinesin construct exhibiting microtubule motor activity contains only the conserved motor domain and neck-linker, although the efficiency with which ATP hydrolysis is coupled to microtubule movement declines dramatically with increasing truncation [Stewart et al. 1993]. These observations clearly support the absolute requirement of a flexible neck-linker for any kind of motility and not just for processive movement. Hence, this crosslinking study confirms the neck-linker as the essential transducer element, but it does not give a direct clue on the validity of the neck- linker unzippering model.
Some results of this crosslinking study seem to conflict with certain aspects of the model. First, the low microtubule affinity of the construct with the fully docked neck-linker was unexpected. In wild-type kinesin nucleotide state and microtubule affinity are mutually dependent, as was shown also for the fully docked neck-linker mutant, although its microtubule affinity was reduced dramatically. Kinesin binds tightly to microtubules only when no nucleotide or ATP is bound. On the other hand, MT binding is necessary for kinesin to release ADP. The position of the neck-linker is independent of the nucleotide state as long as kinesin is in solution [Sindelar et al. 2002]. Once kinesin is bound to microtubules, the conformation and orientation of the neck-linker and nucleotide state are tightly coupled [Rice
et al. 1999]. Upon ATP binding the neck-linker docks onto the catalytic core, while in the ADP state the neck-linker is mobile. These results predict that in the docked neck-linker state (with bound ATP), kinesin exhibits tight microtubule binding. However, the construct with the fully docked neck-linker exhibited very weak microtubule binding (4.4.2.2). It is unclear whether the crosslinked construct does indeed contain ATP in its nucleotide pocket. As the ADP release is not activated by microtubules, probably ADP is bound predominantly to the crosslinked protein, preventing tight microtubule binding. Nevertheless, the conflict remains that even in the AMP-PNP state microtubule affininty is weak. This implies that docking of the neck-linker generally weakens the interactions with microtubules instead of resulting in a tight binding state. These contradictory observations can be reconciled only by the assumption that for the initial weak microtubule encounter leading to ADP release an undocked neck-linker is a prerequesite, while docking of the neck-linker is not an obstacle in the following tight binding state. Hence, this would mean that for initiation and continuation of tight binding different positions of the neck-linker are mandatory.
The second apparent discrepancy relates to the dramatically reduced ATP turnover in the absence of microtubules observed also for the fully docked neck-linker construct (4.4.2.2). Electron paramagnetic resonance analyses showed that the neck-linker freely exchanges between docked and disordered conformations when microtubules are absent [Sindelar et al.
2002]. The conformational equilibrium of the neck-linker is not affected by nucleotide exchange. But this does not allow to conclude the opposite. If nucleotide exchange does not define the position of the neck-linker, its immobilisation may certainly hinder nucleotide exchange. A second possibility how these observations may fit is that in the crosslinked mutant, inhibition of either hydrolysis itself or phosphate release leads to the low ATP turnover, leaving nucleotide exchange unaffected by the position of the neck-linker.