Kinesin-3 motors are fine-tuned at the molecular level to endow distinct mechanical outputs

Abstract

Background Kinesin-3 family motors drive diverse cellular processes and have significant clinical importance. The ATPase cycle is integral to the processive motility of kinesin motors to drive long-distance intracellular transport. Our previous work has demonstrated that kinesin-3 motors are fast and superprocessive with high microtubule affinity. However, chemomechanics of these motors remain poorly understood. Results We purified kinesin-3 motors using the Sf9-baculovirus expression system and demonstrated that their motility properties are on par with the motors expressed in mammalian cells. Using biochemical analysis, we show for the first time that kinesin-3 motors exhibited high ATP turnover rates, which is 1.3- to threefold higher compared to the well-studied kinesin-1 motor. Remarkably, these ATPase rates correlate to their stepping rate, suggesting a tight coupling between chemical and mechanical cycles. Intriguingly, kinesin-3 velocities (KIF1A > KIF13A > KIF13B > KIF16B) show an inverse correlation with their microtubule-binding affinities (KIF1A < KIF13A < KIF13B < KIF16B). We demonstrate that this differential microtubule-binding affinity is largely contributed by the positively charged residues in loop8 of the kinesin-3 motor domain. Furthermore, microtubule gliding and cellular expression studies displayed significant microtubule bending that is influenced by the positively charged insert in the motor domain, K-loop, a hallmark of kinesin-3 family. Conclusions Together, we propose that a fine balance between the rate of ATP hydrolysis and microtubule affinity endows kinesin-3 motors with distinct mechanical outputs. The K-loop, a positively charged insert in the loop12 of the kinesin-3 motor domain promotes microtubule bending, an interesting phenomenon often observed in cells, which requires further investigation to understand its cellular and physiological significance.

KIF1A neurodegenerative disease mutations modulate motor motility and force generation

“…The WT motor showed a mean microtubule gliding velocity of 1.28 µms -1 (Fig. 5, B and G; Movie 1; Table 2), consistent with previous studies (Soppina et al, 2022a;Soppina et al, 2022c). However, gliding velocity of V144F and E233D mutants were found to be 0.42 µms -1 and 0.82 µms -1 , respectively, which is significantly lower than WT motors (Fig.…”

“…Thus, we employed a multi-motor gliding assay to provide insights into the effect of disease mutations on the collective motion of microtubules driven by multiple WT or mutant motors. In the gliding assay, microtubules propelled over a lawn of motor proteins coated onto the cover glass as described (Soppina et al, 2022a;Soppina et al, 2022c). Briefly, adsorb the flow chamber with GFP nanobodies followed by mCitrine-tagged motors to coat the motors on the surface.…”

“…Unlike kinesin-1, KIF1A exhibits inherently fast and superprocessive motility with high affinity for the microtubule (Soppina et al, 2022a;Soppina et al, 2022c;. Mutations in the KIF1A motor are observed in multiple neurodegenerative diseases like Hereditary sensory autonomic neuropathy type-2 (HSAN II) and Hereditary spastic paraplegia (HSP), particularly SPG30.…”

The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau

“…This article contains supporting information ( 6 , 33 , 52 , 54 , 55 , 59 , 61 , 63 , 65 , 84 , 85 , 92 , 93 , 94 , 95 , 96 , 97 ).…”

Editorial: Microtubule-associated molecular motors: Transport mechanisms and role in disease

“…More recently, regulatory factors and regulatory mechanisms of the motor proteins have also become fascinating topics in the motor field. Abnormalities in cytoskeletal elements, defects or alterations in their functions/regulation are often associated with diseases that include neurodegenerative and developmental diseases, cancer and ciliopathies ( Hirokawa et al, 2010 ; Soppina et al, 2014 ; Soppina et al, 2022a ; Soppina et al, 2022b ). Yet, the molecular basis of these pathologies has not been fully understood.…”