The Mechanism of Action of Molecular Motors

R. A. Milligan

Movement is one of the defining features of life. In eukaryotes, movements such as cell contractions, vesicle and organelle translocations and ciliary beating are brought about by systems of molecular tracks and motors. Two classes of tracks have been identified - actin filaments and microtubules. Motors of the myosin superfamily interact with and move along f-actin. Motors of the kinesin and dynein superfamilies interact with and move along microtubules. A few basic concepts underlie the action of all three types of motor molecule: movement of the molecule along the track involves repeated cycles of motor domain attachment, force generation and detachment; this cycle of interaction is coupled to ATP hydrolysis; movements are regulated by accessory proteins acting on either the track or the motor.

Fundamental requirements for understanding the mechanisms of motility in cells are detailed descriptions not only of the atomic structures of the molecules involved, but also of the way in which they interact at various stages during their cycles. Clearly, the first requirement is best satisfied by x-ray crystallography. The high resolution structures of the actin monomer, the myosin head domain and the kinesin motor domain have provided a wealth of information on the mechanism of nucleotide hydrolysis and the remarkable similarity of the underlying protein fold in myosin and kinesin motor domains. The second requirement - that of describing the way in which the proteins interact - has so far proved to be impossible to approach by x-ray crystallography. To achieve this goal, cryo-electron microscopy coupled with image analysis is the method of choice. This approach has proved capable of revealing the detailed micro-anatomy of track-motor complexes and has been essential for understanding the structure of the rigor and ADP-containing states in actomyosin. Thus the two complementary approaches - x-ray crystallography of the individual molecules and cryo-EM and image analysis of the working complexes - are necessary to provide a complete structural understanding of track-motor interactions. In combination with the large and increasing body of biochemical and mechanical data on motors and the current molecular biological armamentarium, the goal of understanding movement production by myosin, kinesin and dynein motors is within reach.

Work in my laboratory currently focuses on members of the myosin and kinesin superfamilies of motors. Our approach is to use cryo-EM and image analysis to calculate three-dimensional (3D) maps of the track-motor complexes at various stages in their movement cycles. The raw 3D maps provide the macromolecular envelopes of the complexes. Individual parts of the complexes such as discrete domains or light chains, are located within the envelopes by difference mapping. For example, a 3D map of myosin lacking a light chain is subtracted from an equivalent "wild type" map. The light chain is visualized as a density peak in the resulting difference map. In a similar fashion, specific amino acid residues are located in the molecular components by gold-cluster labeling and difference mapping. These three types of data - the macromolecular envelopes, the 3D locations of the component parts and the 3D locations of specific amino acid residues - enable us to build near-atomic models of the complexes from the x-ray crystal structures of the individual molecules.

Ron Milligan
The Scripps Research Institute
Department of Cell Biology
10550 North Torrey Pines Road
La Jolla, CA 92037
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