Why a heterotrimer? Elucidating the evolutionary persistence of kinesin-2 through the characterization of its mechanochemical cycle.

Abstract

Molecular motors play important in many critical cellular functions. Here I describe three molecular motor families: DNA polymerase, Myosin and Kinesin. Each motor has developed a unique approach to increasing their time spent interacting with their respective tracks, which is called processivity. This increase in processivity allows these motors to perform their respective jobs accurately and efficiently. I look deeper into the structures that make up the kinesin family of motor proteins to better understand the relationship between the motor and the track, and how the relationship tunes processivity.

The kinesin family of motor proteins relies heavily on the homodimeric architecture for the assembly of motor proteins; however, heterotrimeric KIF3AB has been shown to be as processive as kinesin-1 while having different mechanochemical properties. This leads to the question: "Why is KIF3AB a heterotrimer?" The non-redundant cellular functions could hint at the advantages required from the motor proteins. Different subsets of microtubules are present in cilia, and the functional differences between the motor head allows for two different motor heads that could associate differently with these microtubules. Our data supports these differences in association by indicating that KIF3AB has a bias for the KIF3A motor domain. Phosphate release kinetics confirms that the initial association and release of phosphate occurs from the KIF3A motor domain. Finally, the specificity for binding the scaffold protein, kinesin associated protein (KAP) could be the reason for the heterotrimer. The associated tails of the KIF3A and KIF3B polypeptide could be required for binding to KAP subsequently providing the scaffold for intraflagellar transport.

We have constructed truncated murine KIF3AB, KIF3AA, and KIF3BB that purify in high yields from bacteria and are well-behaved in our assays. We pursued a presteady-state kinetic analysis of the ATPase mechanism and coordination of KIF3A and KIF3B stepping, which will better define the key principles that underlie KIF3AB's processivity in comparison to homodimeric processive kinesins. We found that KIF3AB has a rate-limiting step of ADP release upon initial collision at 13 s-1. Measurements of the presteady-state kinetics reveal that dissociation that is tightly coupled to Pi release about 20 s-1 could be the slow step that occurs during processive stepping.

Conventional kinesin along with KIF3AB are described in detail. These motors share a common kinesin motor domain however they do not function in the same cellular context. KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. Primarily working with cytoplasmic dynein, KIF3AB is required for the assembly and maintenance of cilia and flagella. There has been significant interest in KIF3AB in examining its unique heterodimeric architecture. Investigations have focused on KIF3AB's tightly associated neck coiled-coil, lengthened neck linker, and unique stalk formation. Studies have identified structural features that are well suited for transporting cargo through the highly crowded cilia and flagella. However, low purification yields of well-behaved KIF3AB have hampered detailed kinetic studies.

To further investigate the contribution of each motor domain, forced homodimeric KIF3AA and KIF3BB motor proteins were examined using presteady-state kinetics. The results reveal similar mantADP release kinetics between KIF3AA and KIF3AB. The phosphate release kinetics of the motors also reveals that KIF3AA and KIF3AB share a similar rate of release. These data indicate that KIF3AB could have a bias that allows for the KIF3A motor domain to interact with the microtubule first.