Identification of a microtubule-binding domain in a cytoplasmic dynein heavy chain.
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 functional domains of a dynein heavy chain from Dictyostelium. The carboxyl-terminal 10-kilobase region of the heavy chain encodes a 380-kDa polypeptide that approximates the globular head domain. Attempts to further truncate this region fail to produce polypeptides that either bind microtubules or UV-vanadate cleave, indicating that the entire 10-kilobase fragment is necessary to produce a properly folded 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 resulting head domain polypeptide no longer binds microtubules; when the excised region is expressed in vitro, it cosediments with 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 with microtubules is complex.
Koonce
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Division of Molecular Medicine, Wadsworth Center, Empire State Plaza, Albany, New York 12201-0509, USA. Michael.Koonce@wadsworth.org
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003593:Cytoplasm / Q000201:enzymology; D004023:Dictyostelium; D004398:Dyneins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D017384:Sequence Deletion; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014638:Vanadates / Q000378:metabolism
D014638:Vanadates; D000255:Adenosine Triphosphate; D004398:Dyneins