Elucidation of the fundamental working mechanism of heteromeric kinesins via single molecule optical trapping and single fluorophore localization
Final Report Abstract
In our work, we presented insights into the fundamental mechanistic properties of KLP11/KLP20 and provided a biological rationale for heterodimerization. A motif in the KLP11/KLP20 tail is identified that when mutated leads to a constitutively active motor in vitro. This auto-inhibitory switch only functions when the motor heads are in their correct wild type positions. Swapping the relative positions of the motor domains abolishes this auto-regulation as shown by the processive and but unregulated chimeric kinesin. To further dissect the contribution of the individual heads to the overall processivity in the wild type KLP11/KLP20 motor, chimeras containing two identical heads were expressed with motor heads of KLP11 or KLP20 with their neck linkers fused to their heterodimeric tails to ensure reliable dimerization. The most intriguing finding was that KLP11 is mechanically unprocessive as a homodimer, meaning that as a single motor it is not capable of supporting appreciable run lengths as expected of a motor that builds and maintains several micronlong cilia. Remarkably, the same subunit yields a robustly processive motor when dimerized with its partner KLP20. The KLP11/KLP20 heterodimeric kinsin-2 thus extends prior models of processivity that were based solely on studies of native homodimeric motor proteins. In summary, a detailed mechanistic analysis presented in this work demonstrated the first example of a motor protein in nature that combines a processive motor domain with an unprocessive one to take robust processive steps. Furthermore, the quantitative analysis revealed that the wild type heterodimeric KLP11/KLP20 motor is regulated asymmetrically by its unprocessive head domain. This also represents a novel way of how motor proteins can be autoinhibited in addition to what has been known so far from native homodimeric motors.
