Long Range Allosteric Control of Cytoplasmic Dynein ATPase Activity by the Stalk and C-terminal Domains

Abstract

The dynein motor domain consists of a ring of six AAA domains with a protruding microtubule-binding stalk and a C-terminal domain of unknown function. To understand how conformational information is communicated within this complex structure, we produced a series of recombinant and proteolytic rat motor domain fragments, which we analyzed enzymatically. A recombinant 210-kDa half-motor domain fragment surprisingly exhibited a 6-fold higher steady state ATPase activity than a 380-kDa complete motor domain fragment. The increased ATPase activity was associated with a complete loss of sensitivity to inhibition by vanadate and an ϳ100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated ϳ1000-fold by microtubule binding to the 380-kDa motor domain. Both the half-motor and full motor domain fragments were remarkably resistant to tryptic proteolysis, exhibiting either two or three major cleavage sites. Cleavage near the C terminus of the 380-kDa motor domain released a 32-kDa fragment and abolished sensitivity to vanadate. Cleavage at this site was insensitive to ATP or 5-adenylyl-␤,␥-imidodiphosphate but was blocked by ADP-AlF 3 or ADP-vanadate. Based on these data, we proposed a model for long range allosteric control of product release at AAA1 and AAA3 through the microtubule-binding stalk and the C-terminal domain, the latter of which may interact with AAA1 to close the motor domain ring in a cross-bridge cycle-dependent manner.Cytoplasmic dynein is a microtubule-based motor that utilizes energy from ATP hydrolysis to facilitate a wide variety of functions in eukaryotic cells such as spindle alignment, nucleus positioning and chromosome separation during mitosis, and retrograde axonal transport of subcellular components (1, 2). Dynein belongs to the functionally diverse family of AAAϩ proteins (3). Members of this family contain a conserved domain of 200 -250 residues termed AAA, or "ATPases associated with various cellular activities," that contains five signature motifs involved in ATP binding and hydrolysis. Most AAA proteins form homohexameric rings in which each subunit is composed of either one or two AAA domains. The ϳ380-kDa dynein motor, in contrast, consists of a ring of six dissimilar and covalently linked AAA domains and at the extreme C terminus an ϳ30-kDa region of unknown function. Fused to the motor domain is a substantial projection, referred to as the stem, which contains sites for dimerization and association with cargo binding subunits (4). Emerging from between AAA4 and AAA5 is another projection, the stalk, which is thought to consist of an antiparallel coiled-coil with a globular domain for microtubule binding at its tip (5).Sequence analysis of the dynein motor domain suggests that the ability to bind and hydrolyze nucleotides differs among the six AAA domains (6). Whereas the first AAA domain contains the entire set of nucleotide-interacting motifs, the probability of identifying these motifs in AAA2-AAA4 ...

Kinetic models for the coordinated stepping of cytoplasmic dynein

“…Note that these predictions are tightly coupled to the assumption that following ATP hydrolysis, a head containing both ADP and Pi is detached from ͑or weakly interacts with͒ the MT. Current experimental results 8,14 disagree on this issue. If future experiments confirm the high sensitivity of the motor's velocity and run length on the phosphate concentration, this result would provide indirect evidence to support the scenario in which heads containing both ADP and Pi do not interact strongly with the MT.…”

“…11,14 It has been shown that the motor's interaction with the MT accelerates the release of ADP in both axonemal 11 and cytoplasmic dynein, 14 which suggests that MT binding precedes ADP release.…”

The dynein family at a glance

“…AAA1 has been deduced to serve as the principal site for ATP hydrolysis, based on the effects of vanadate-mediated UV photocleavage at this site (Gibbons et al, 1987;Gee et al, 1997). More recent studies on the dynein motor domain suggest that hydrolytic activity in AAA1 also requires the structural involvement of AAA2 (Takahashi et al, 2004;Höök et al, 2005). Consequently, more than half of the conserved residues are located within the boundaries of the linker and the first two AAA domains, stressing the region's significance as the source of force production within the dynein motor.…”

Autoinhibitory and other autoregulatory elements within the dynein motor domain

“…Our lab has recently investigated intramolecular regulation of dynein enzymatic activity by analysis of recombinant and proteolytic motor fragments (Höök et al, 2005). Using baculovirus infection of insect cells, we expressed a 380-kDa fragment corresponding to the entire motor domain and a 210-kDa fragment corresponding to its Nterminal half, ending just prior to the stalk (Fig.…”

“…To learn more about the structural organization and intramolecular regulation of the dynein motor domain, we subjected both the 380-and 210-kDa fragments to controlled proteolytic digestion (Höök et al, 2005). Surprisingly, the number of discrete fragments that could be identiWed was very small.…”