Feedback of the Kinesin-1 Neck-linker Position on the Catalytic Site

Abstract

Kinesin-1 motor proteins step along microtubules by a mechanism in which the heads cycle through microtubule-bound and unbound states in an interlaced fashion. An important contribution to head-head coordination arises from the action of the neck-linker that docks onto the core motor domain upon ATP binding. We show here that the docked neck-linker not only guides the microtubule-unbound head to the next microtubule binding site but also signals its position to the head to which it is attached. Cross-linking studies on mutated kinesin constructs reveal that residues at the interface motor core/docked neck-linker, among them most importantly a conserved tyrosine, are involved in this feedback. The primary effect of the docked neck-linker is a reduced microtubule binding affinity in the ADP state.Kinesin-1 (formerly conventional kinesin) proteins are molecular motors that move along microtubules in a stepwise fashion at the expense of ATP hydrolysis energy. A large body of evidence shows that these motors are dimeric and generate motility by the alternating action of two identical motor domains (1-6). A crucial structural intermediate is a Kinesin-1 molecule that is strongly attached to the filament by one nucleotide-free head, whereas the other one remains in a weakly microtubule-binding ADP state. Upon ATP binding, a very rapid structural change of the neck-linker of the microtubule-bound motor head occurs that allows the partner head to lose its ADP and to bind tightly to the microtubule (7). The neck-linker, a short stretch of ϳ10 amino acid residues that follows immediately C-terminal of the catalytic core motor domain, is thought to exist in at least two different conformations, one that is docked or zippered to the motor core, and a flexible undocked structure (8, 9).The docked neck-linker is visible in some crystal structures (10, 11), and its path along microtubule-bound kinesin has been deduced from cryo-EM (8, 12). According to these data, the neck-linker zippers along a groove in the core motor domain between helix 6 on one end, and loop 10 on the other end where it is held by a meshwork of hydrogen bonds. The nature of the undocked conformation is not clear and might differ in truncated, monomeric constructs (that have been used for many of the structural investigations) and dimeric ones.The conformational change of the neck-linker has been used to explain the mechanism of Kinesin-1 motility. It is thought that necklinker docking allows or causes the second head to bind to a forward microtubule binding site. Interestingly, it is unlikely that the docking process of the neck-linker generates the force exerted by kinesin. The free energy of neck-linker docking is surprisingly low (⌬G ϳϪ1 kJ/mol; Ref. 9), and hardly accounts for the force that can be generated by a single kinesin molecule (5-6 pN, Ref. 13). Estimated from crystal structures, the length of the neck-linker is at least 4 nm. Accordingly, ⌬G will be Ն 12 kJ/mol, which is roughly 10-fold larger than the measured free enthalpy change of neck-l...

Molecular origin of the weak susceptibility of kinesin velocity to loads and its relation to the collective behavior of kinesins

“…It is in accordance with an experimental study showing that both the neck linker and ATP bind to a motor head (31). This coupling between the neck linker and the ATP binding site was also observed by other research groups (32,33). In this work, we further identified how this coupling was regulated by external loading forces.…”

CK2 activates kinesin via induction of a conformational change

“…However, there are likely other conformations of the NL that position it close to the MD but that are different from this docked state that has been visualized by ATP-like crystal structures. One such conformation was previously induced by selective cross-linking, which artificially induced NL immobilization (via designed disulfide bonds) and led to the complete inhibition of kinetic activity for monomeric as well as dimeric kinesin constructs (17). Our inactivation-with concurrent loss of microtubule binding-could thus be similar to the decreased microtubule affinity observed previously when the NL was artificially held in place close to the MD (17).…”

“…Instead, we hypothesize that a smaller change occurs. Recent work highlighted the position of the NL domain relative to the rest of the head as changing during the enzymatic cycle and as being important for alteration of the MT binding affinity (16,17). Thus, kinesin inactivation might reflect a conformational change that alters the position/conformation of the NL relative to the head.…”

“…One such conformation was previously induced by selective cross-linking, which artificially induced NL immobilization (via designed disulfide bonds) and led to the complete inhibition of kinetic activity for monomeric as well as dimeric kinesin constructs (17). Our inactivation-with concurrent loss of microtubule binding-could thus be similar to the decreased microtubule affinity observed previously when the NL was artificially held in place close to the MD (17). Thus, one interpretation of our data is that the NL is not truly "docked" but, rather, is adsorbed onto the MD in a different "off-pathway" conformation, reminiscent of the positioning of the Eg5 NL in the ADP-bound state (19).…”

Single molecule mechanics of the kinesin neck

“…Many members of the kinesin and myosin motor families are double-headed with the 2 heads joined by a double-helical coiled coil. In the case of the molecular motor kinesin, various studies have shown that for processive movement along microtubular tracks, a communication between the 2 heads through mechanical strain is essential (1)(2)(3)(4). A recent study by Yildiz et al (5) has shown that transmission of the strain generated by neck-linker docking can be critically affected by inserting artificial peptides between neck-linker and neck, thus introducing slack into the connection between the 2 heads.…”