The role of multiple ATP-binding sites in dynein motor domain

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Dynein is a huge complex molecule including heavy chain(s) which have multiple (4) ATP binding consensus sequences (P-loops) as well as intermediate and light chains. We purified a monomeric form of dynein (dynein-a) from 14S dyneins of Tetrahymena thermophila and characterized it. In in vitro motility assays, dynein-a rotated microtubules around their longitudinal axis as well as translocated them with their plus-ends leading. ATPase activity at 1 mM ATP was doubled in the presence of a low level of ADP. Both ATPase activity and transnational velocities in the presence of ADP fit the Michaelis- Menten equation well. However in the absence of ADP, neither of the activities followed the Michaelis-Menten-type kinetics probably due to the effect of two-ATP binding sites. Our results also indicate that dynein-a has an ATP-binding site that is very sensitive to ADP and affects ATP hydrolysis at the catalytic site. This study shows that a monomeric form of dynein molecule regulates its activity by direct binding of ATP and ADP to itself, and thus the dynein molecule has an intramolecular regulating system.