The structural cycle of kinesins at atomic resolution: kinesin-tubulin interaction and nucleotide state. – (Kines-Tubul-DARP)-in

Cytoskeletal motors are integral to all forms of eukaryotic life, with roles ranging from intracellular transport to cell division. They use the energy released upon ATP hydrolysis to produce work and most often move either along actin filaments, in the case of myosins, or along microtubules for dyneins and kinesins. There is a tight coupling between the nucleotide cycle (binding, hydrolysis and release) and the structural changes at the basis of the motor mechanism. Atomic structures of functional motor domains of all classes of cytoskeletal motors (kinesin, dynein and myosin) detached from their respective filaments are known. Besides, electron microscopy has provided low resolution models of decorated filaments. So far, structural methods have not yielded atomic-resolution descriptions of the motor-filament complexes. The biochemical properties of cytoskeletal motors change considerably upon interaction with their cognate filament. Consequently, available structural models do not account for many essential properties of the cytoskeletal motors.
Our main goal is to establish the first complete structural cycle for a cytoskeletal motor by determining by X-ray crystallography the atomic structure of motile kinesin functional motor domains in complex with tubulin, the microtubule building block, along with the kinesin nucleotide cycle. We will also study microtubule depolymerising kinesins.
To reach this objective, this proposal relies on the development of tubulin sequestering tools, which are in fact ‘chaperones’ for crystallization. We have selected anti-tubulin DARPins in the course of the ‘mec-tub’ ANR project. Whereas these DARPins have already proven useful and are being used as such, their affinities for tubulin (Kd in the 0.1 µM range) are limiting in some cases. Our first step serves as a basis for the whole project. We will screen available DARPins to identify those that do not interfere with kinesins for tubulin binding and evolve the selected ones towards higher affinities.
The resulting DARPins will be submitted to crystallization experiments as ternary complexes with tubulin and functional motor domains of several motile kinesins, in their three main nucleotide states (ATP, ADP and nucleotide free) as well as bound to a transition-state analogue of nucleotide hydrolysis (ADP-AlFx). Our preliminary results clearly demonstrate this is possible. This will fulfil our main goal.
A second objective is the determination of the structural mechanism of a microtubule-depolymerising kinesin complex. These kinesins, in the kinesin-13 family, target microtubule ends and induce disassembly instead of walking along microtubules. Their nucleotide cycle, adapted to their function, differs from that of motile kinesins. In particular, they interact with tubulin only when they are ATP-bound whereas motile kinesins bind to tubulin in the ATP- and nucleotide-free states. In this case, the main target is the structure of a kinesin-13(ATP state)-tubulin complex. We have expressed functional domains of the three human class 13 kinesins but, as they all have similar physico-chemical properties, we also plan to study more divergent ones, e.g. from diatoms. These kinesins will be characterized biochemically to ascertain their depolymerase activity and, in case this is indeed verified, structurally.