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Structure of Molecular Tracks and Motors

R.A. Milligan, B.O. Carragher,* H. Celia, D.P. Dias, J.D. Jontes, T.T. Pham, M. Whittaker, E.M. Wilson-Kubalek

* Beckman Institute, Urbana, IL

Our research focuses on the mechanism of chemomechanical transduction by myosin and kinesin motor proteins. Our goal is to visualize and describe the conformational changes in the track-motor complexes that occur during the ATP hydrolysis cycle. The tracks are linear polymers, and neither they nor the track-motor complexes are amenable to x-ray crystallographic analysis. However, some of the separate components--the myosin head, the actin monomer, and domains from 2 kinesin-like motors--have been crystallized, and atomic models of the proteins are available.

We use cryo-electron microscopy and image analysis to calculate 20-Å-resolution 3-dimensional maps of the track-motor complexes in the presence and absence of nucleotides and nucleotide analogs. These maps of the entire complex are used together with the x-ray structures of the individual components 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 (nucleotide-free) complex and have visualized a dramatically different conformation of smooth muscle myosin II, nonmuscle myosin IIB, brain myosin V (in collaboration with L. Sweeney, University of Pennsylvania), and brush border myosin I heads bound to actin in the presence of ADP. ADP has little effect on the interaction geometry of the myosin motor domain with actin. However, the myosin domain that contains the 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. Current work is aimed at exploring the generality of the ADP-induced changes and at visualizing earlier stages in the cross-bridge cycle. With these experiments, we expect to determine the relative contributions of the various biochemical steps to the power stroke in a variety of myosins.

Additional work on actomyosin focuses on myosin-based regulatory mechanisms. We are using cryo-electron microscopy, image analysis, and model building approaches to investigate the structural basis of regulation by light-chain phosphorylation (smooth muscle myosin II), heavy-chain phosphorylation (myosin I), and calmodulin association-dissociation (brush border myosin I and myosin V).

In collaboration with R. Vale, University of California, San Francisco, we are using the same general approaches to study the attachment of kinesin motors to their microtubule tracks. We showed that kinesin and ncd motor domains bind to microtubule protofilaments with the same geometry of interaction, despite the opposite direction of movement of the molecules. We have docked the x-ray structure of the kinesin motor domain into 3-dimensional electron image maps of the microtubule-motor complex and have determined the structural elements of kinesin responsible for microtubule binding. Most recently, we combined 3-dimensional microtubule maps and the high-resolution structure of tubulin to build an atomic model of the microtubule. This work provides clues for understanding track-motor interactions and the structural basis of dynamic instability, cold sensitivity, and microtubule stabilization by paclitaxel (Taxol). Current work focuses on determining the 3-dimensional structure of double-headed motors trapped at various stages in their cycle of interaction with microtubules. These data will provide insights into the mechanism of movement generation and the origin of directionality.

PUBLICATIONS

Jontes, J.D., Milligan, R.A. Brush border myosin-1 structure and ADP-dependent conformational changes revealed by cryo-electron microscopy and image analysis. J. Cell Biol. 139:683, 1997.

Jontes, J.D., Milligan, R.A., Pollard, T.D., Ostap, E.M. Kinetic characterization of brush border myosin-I ATPase. Proc. Natl. Acad. Sci. U.S.A. 94:14332, 1997.

Jontes, J.D., Ostap, E.M., Pollard, T.D., Milligan, R.A. Three-dimensional structure of Acanthamoeba myosin-IB determined by cryo-electron microscopy of decorated actin filaments. J. Cell Biol. 141:155, 1998.

Wilson-Kubalek, E.M., Brown, R.E., Celia, H., Milligan, R.A. Lipid nanotubes as substrates for helical crystallization of macromolecules. Proc. Natl. Acad. Sci. U.S.A. 95:8040, 1998.