Key residue on cytoplasmic dynein for asymmetric unbinding and unidirectional movement along microtubule

Abstract

Cytoplasmic dynein 1 is almost exclusively responsible for intracellular transport toward the minus-end of microtubules in animal cells. One of the key factors for the unidirectional movement of dynein is the asymmetry of the unbinding of the motor from the microtubule when an external load is applied; it dissociates more easily from microtubules with minus-end directed loading than with plus-end directed loading. To elucidate the molecular basis for this property, we performed molecular dynamics simulations to identify the key residues responsible for asymmetry, which were then examined experimentally. First, we reproduced asymmetry in the unbinding behavior of dynein using coarse-grained simulations. Second, data analysis together with mutational analysis in silico predicted the specific residues that may be responsible for the asymmetry in unbinding. Third, to examine this prediction, we expressed and purified recombinant dynein with mutations in either of the identified key residues. Consistent with the simulations, one of the mutants did not exhibit asymmetry in the in vitro binding assay. Moreover, the mutant dynein was able to move diffusely along a microtubule but was unable to restrict its movement to the minus-end direction. Thus, our results demonstrate both experimentally and theoretically how the key residue on the microtubule-binding domain generates asymmetry in unbinding, which is a critical mechanism for the unidirectional movement of dynein along a microtubule track.

Regulatory mechanisms of the dynein-2 motility by post-translational modification revealed by MD simulation

“…The lateral landing of dynein-2 can only be restricted within these two PF lanes. A recent study used the similar coarse grain MD set up to our study to investigate dynein motility under external load 41 . They were able to mutate the key residues found in the simulation and express the mutated dyneins and verify the MD results using in vitro motility assay.…”