What Determines the Directionality of Kinesins? Studying the Anomalous Directionality of Nonclaret Disjunctional...

NCD (nonclaret disjunctional) motors are a class of kinesin family proteins that are known to walk toward the minus end along the microtubule. Understanding the origin of such unique directionality of NCD motors and related kinesins has been a problem of fundamental importance. Experimental studies have indicated that specific NCD motor mutants switch their directionality. A type of NCD mutant (ran12) walks in the opposite direction (plus end) compared to the wild type (WT), whereas another mutant (N340K) of NCD shows diffusive bidirectionality. This work establishes that the directionality of these NCD motors is regulated by the barrier of the rate-determining ADP release step. We explore the molecular origin of the anomalous directionality of the mutated NCD motors compared with the wild type and determine the change in the barriers of ADP release for forward and backward motions. Our investigation is performed by combining different simulation techniques such as coarse-grained (CG) modeling, all-atom steered molecular dynamics (SMD) simulation, free energy estimation, as well as structural and stability analysis, and the maximum entropy (MaxEnt) method. We conclude that the differences in the directionality of different types of NCD motors are controlled by the free energy barrier for the ADP release step. Finally, we show that the statistical energy deduced from homologous sequences by the maximum entropy (MaxEnt) principle is significantly correlated with the stability and experimentally measured velocities of the wild type and several NCD mutants, and established that the stability loss of specific NCD mutants contributes to their reduced velocities and anomalous directionality on the microtubules. This provides a rare case where the origin of the AI prediction is clearly related to a physical-based picture.