Molecular basis of the kinematics of the kinesin step

Abstract

Kinesin is an ATP-dependent cellular transporter that ferries cargos towards the plus-end of a microtubule. Despite significant advances in experiments, which have provided deep insights into the motility of kinesin, the molecular events that occur in a single step have not been fully resolved. In order to provide these details, this thesis develops a structure of the complex between kinesin and microtubule, and devises new simulation methods to probe the stepping kinetics over a wide range of conditions. Hundreds of molecular movies of kinesin walking on the microtubule are generated using coarse-grained simulation methods. Analysis of these movies shows that there are three major stages in the stepping kinetics of kinesin. In addition, an allosteric network within kinesin, responsible for controlling nucleotide release, is identified using microsecond all-atom simulations. These simulations are used to answer two important questions.

First, does kinesin move by a "power stroke" or by diffusion? During a single step, the trailing head of the kinesin detaches from the microtubule, passes the microtubule-bound leading head, and attaches to the target binding site 16 nm away. The target binding site, however, is one of eight accessible binding sites on the microtubule. Is it possible that the "power stroke" (a large conformational change) in the leading head, pulls the trailing head into the neighborhood of the target binding site? This remained unclear because the fraction of the 16 nm step associated with the power stroke and diffusion had never been quantified.

Second, how does the microtubule accelerate ADP release from kinesin, which is a key step in completing a single step? The ADP binding site of kinesin is more than 1.5 nm away from the microtubule binding surface. Therefore, the microtubule must affect the ADP binding site through an allosteric mechanism. However, the structural basis for transmitting signals through the underlying allosteric network was previously unknown.

Analysis of hundreds of kinesin steps generated using coarse-grained simulations showed that the power stroke associated with the docking of the neck linker to the leading head, is responsible for only 4 nm of the 16 nm step, and the remaining 12 nm is covered by diffusion. However, the power stroke in the leading head constrains the diffusion of the trailing head, decreases the probability of side steps, and therefore biases the trailing head, to the target binding site. Additional all-atom simulations of the ADP-kinesin-microtubule complex, revealed a surprisingly simple allosteric network within kinesin that explains the acceleration of ADP release upon microtubule binding. The allosteric network also explains two additional experimental observations on ADP release from kinesin.