News and Publications

Structure of Molecular Tracks and Motors

R.A. Milligan, J.M. Al-Bassam, C.M. Birdsell, S.M. Cain, B.O. Carragher,* A.W.-C. Lin, I. Rouiller, M. Whittaker, E.M. Wilson-Kubalek, W. Wriggers

* Beckman Institute, Urbana, IL

Our research focuses on elucidating the structure-function relationships of myosin and kinesin motor proteins. Our goal is to visualize and describe the conformational changes in the track-motor complexes that occur during the cycles of interaction. We use electron cryo-microscopy and image analysis to calculate 20-Å-resolution 3-dimensional maps of the complexes in the presence and absence of nucleotide analogs. These maps of the entire complexes and the x-ray structures of the individual components are used to build high-resolution models of the working assemblies. In this way, we obtain a detailed picture of how the motors interact with their tracks at various stages in the chemomechanical cycle.

So far we have built models of the actomyosin rigor complex and have visualized dramatically different conformations of smooth muscle myosin II, nonmuscle myosin IIB, brain myosin V, myosin VI, and brush border myosin I heads bound to actin in the presence of ADP. Most of this work is being done in collaboration with H.L. Sweeney, University of Pennsylvania. We found that ADP has little effect on the interaction geometry of the myosin motor domain with actin. However, the myosin domain that contains light chains undergoes a dramatic change in orientation when this nucleotide is present. Our data suggest that the later stages of the ATPase cycle, which culminate in release of ADP, contribute to the power stroke in these myosin motors. Most interestingly, we found that myosin VI has an unusual ADP-induced movement, suggesting that this myosin moves in the opposite direction to all other members of the myosin superfamily. Additional experiments are under way to explore this result.

In collaboration with R. Vale, University of California, San Francisco, we are using the same general approach to study the attachment of kinesin motors to microtubules. We showed that brain kinesin and ncd motor domains bind to microtubule protofilaments with the same geometry of interaction, despite the opposite direction of movement of these molecules. This result focused attention on the neck-linker region of these molecules. Most recently, we completed a study in which gold cluster labels were attached to the neck-linker of motor molecules. This gold-labeling approach enabled us to indirectly visualize the orientation of the neck-linker at 4 well-defined stages in the ATPase cycle. The findings allow us to propose a mechanochemical cycle for kinesin motors.

Exciting technical advances in our group include the development of a general approach for helical crystallization of macromolecules on tube-forming lipids and the implementation of automatic grid searching and image acquisition protocols for electron cryo-microscopy.

PUBLICATIONS

Celia, H., Wilson-Kubalek, E., Milligan, R.A., Teyton, L. Structure and function of a membrane-bound murine major histocompatibility complex class I molecule. Proc. Natl. Acad. Sci. U. S. A. 96:5634, 1999.

Dias, D.P., Milligan, R.A. Motor protein decoration of microtubules grown in high salt reveals the presence of mixed lattices. J. Mol. Biol. 287:287, 1999.

Nogales, E., Whittaker, M., Milligan, R.A., Downing, K.H. High-resolution model of the microtubule. Cell 96:79, 1999.

Oegema, K., Wiese, C., Martin, O.C., Milligan, R.A., Iwamatsu, A., Mitchison, T.J., Zheng, Y. Characterization of two related Drosophila l-tubulin complexes that differ in their ability to nucleate microtubules. J. Cell Biol. 144:721, 1999.

Viegel, C., Coluccio, L.M., Jontes, J.D., Sparrow, J.C., Milligan, R.A., Molloy, J.E. The motor protein myosin-1 produces its working stroke in two steps. Nature 398:530, 1999.

Wriggers, W., Milligan, R.A., McCammon, J.A. Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. J. Struct. Biol. 125:185, 1999.

Wriggers, W., Milligan, R.A., Schulten, K., McCammon, J.A. Self-organizing neural networks bridge the biomolecular resolution gap. J. Mol. Biol. 284:1247, 1998.