Neck-linker And Neck-coil both contribute to Kinesin Processivity

Abstract

Kinesins are microtubule-based molecular motors involved in intracellular transport. These motors utilize energy from ATP hydrolysis to ‘walk’ along microtubules and are processive, meaning that they take multiple steps before detaching from the microtubule. The kinesin superfamily consists of 14 families of motors that have diverse structures and diverse cellular functions. A principle question in understanding motor function is: What are the structural determinants of processivity. In contrast to homodimeric motors in the canonical Kinesin-1 family, Kinesin-2 motors have two different head domains and a three amino acid extension in their neck linker. We showed previously that Kinesin-2 motors are slower than Kinesin-1 and are roughly four-fold less processive. These differences could result from biochemical differences in the head domains, reduced inter-head coordination due to differences in the neck-linker domains, or diminished electrostatic interactions between the coiled-coil domain and the microtubule track. To test the influence of the coiled-coil domain, we engineered motors containing the Kinesin-2 head and neck linker domains fused to the Kinesin-1 coiled-coil. Single-molecule fluorescence experiments of GFP-labeled motors showed enhanced processivity compared to Kinesin-2, indicating a role for the coiled-coil in motor processivity. When the Kinesin-1 neck-linker domain was extended by three amino acids, processivity fell by a factor of three, suggesting that the neck-linker domain is an important determinant of processivity. However, shortening the Kinesin-2 neck-linker significantly reduced its processivity, indicating that in a given motor, the head, neck-linker and coiled-coil domains are tuned for optimal motor function.