IDENTIFICATION OF A MICROTUBULE-BINDING DOMAIN IN A CYTOPLASMIC DYNEIN HEAVY-CHAIN

As a molecular motor, dynein must coordinate ATP hydrolysis with confo rmational changes that lead to processive interactions with a microtub ule and generate force. To understand how these processes occur, we ha ve begun to map functional domains of a dynein heavy chain from Dictyo stelium. The carboxyl-terminal 10-kilobase region of the heavy chain e ncodes a 380-kDa polypeptide that approximates the globular head domai n. Attempts to further truncate this region fail to produce polypeptid es that either bind microtubules or UV-vanadate cleave, indicating tha t the entire 10-kilobase fragment is necessary to produce a properly f olded functional dynein head. We have further identified a region just downstream from the fourth P-loop that appears to constitute at least part of the microtubule-binding domain (amino acids 3182-3818). When deleted, the resulted head domain polypeptide no longer binds microtub ules; when the excised region is expressed in vitro, it cosediments wi th added tubulin polymer. This microtubule-binding domain falls within an area of the molecule predicted to form extended alpha-helices. At least four discrete sites appear to coordinate activities required to bind the tubulin polymer, indicating that the interaction of dynein wi th microtubules is complex.