Dynein arm conformation and mechanochemical transduction in the eukaryotic flagellum

Abstract

When partially disrupted axonemes of Chlamydomonas were negatively stained in the presence of ATP, the dynein arms appeared club-shaped and were tilted proximally. In the absence of ATP, or in the presence of the non-hydrolysable ATP analogue, adenylyl imidodiphosphate, the arms on most doublets appeared to be made up of a thin diagonal component plus a larger component which resembled the arms seen in the presence of ATP but was more upright. Under these same conditions, the arms on some doublets appeared as distally directed ellipses. The two different images observed in the absence of ATP were interconvertible by tilting the doublets in a goniometer stage, indicating that they represented arms in the same conformational state viewed from two different angles. A three-dimensional model has been constructed which accounts for the appearance of the arms at the different angles. Tilting did not convert either of these images into those seen in the presence of ATP. Examination of mutants lacking the inner and outer arms indicated that all of the images were derived from the outer arm. In the absence of ATP, arms were frequently observed which were bound to a B tubule but had lost their attachment to the A tubule; such arms were not observed in the presence of ATP. These observations provide direct evidence for an ATP-dependent conformational change in the outer arm, and suggest a sequence of steps for the mechanochemical cycle of this arm.