STRUCTURE-FUNCTION RELATIONSHIPS OF A DYNEIN ATPASE

M.P. Koonce, M. Samso, M. Radermacher, J. Frank, and I. Tikhonenko
Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany NY 12201-0509

As a molecular motor, dynein must coordinate ATP-hydrolysis with conformational changes that lead to processive interactions with a microtubule and generate force. To understand how these processes occur, we have begun to map structure-function relationships of a dynein heavy chain (DHC) from Dictyostelium. The carboxy-terminal 10 kb region of the DHC encodes a 380 kDa polypeptide that approximates the globular head domain. TEM and computer image processing have been used to produce 2D averages and low resolution 3D reconstructions of negatively-stained dynein motor domains. These images show an oblong round structure of 12 - 13 nm in diameter, consisting of a central stain-filled area surrounded by 7-8 radial globular regions. Attempts to further truncate the DHC fail to produce polypeptides that either bind microtubules or UV-vanadate cleave, indicating that this entire 380 kDa fragment is necessary to produce a properly folded, functional dynein head. We have identified a region just downstream from the fourth P-loop that appears to contain dynein's microtubule-binding activity (aa 3182 - 3818). When deleted, the resulting polypeptide no longer binds microtubules; when the excised region is expressed in vitro, it cosediments with tubulin polymer. This microtubule-binding domain falls within an area of the molecule predicted to form extended a-helices. At least four discrete sites appear to coordinate activities required to bind the tubulin polymer, indicating that dynein's interaction with microtubules is complex. Currently we are trying to relate this functional activity to the structural information obtained by electron microscopy.

Supported in part by the NIH, NSF, and a Fellowship from the Ministry of Education and Science, Spain.