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

Peter Höök,Atsushi Mikami,Beth Shafer,Brian T Chait,Steven S Rosenfeld,Richard B Vallee

doi:10.1074/jbc.m504693200

Peter Höök, Atsushi Mikami + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m504693200

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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 approximately 100-fold increase in the rate of ADP release. The time course of product release was discovered to be biphasic, and each phase was stimulated approximately 1000-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-beta,gamma-imidodiphosphate but was blocked by ADP-AlF3 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.

Highlights

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]
Recent work on P-loop mutants showed that nucleotide binding at the first and third AAA domains is required for ATPinduced release of microtubules [11] and for microtubule stimulation of ATPase activity [8], supporting long range allosteric communication through unknown conformational changes within the motor domain
To understand the mechanism by which production of force is coupled with ATP hydrolysis within the dynein motor, we produced a series of recombinant and proteolytic motor domain fragments, and we determined their enzymatic properties

Summary

Introduction

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. The first fragment corresponded to the entire 380-kDa motor domain, and the second to the N-terminal 210-kDa half of the motor domain (half-motor domain) Both fragments contain the entire machinery for ATP hydrolysis, they unexpectedly display distinctive enzymatic properties. Based on our findings we suggest a model in which both the stalk and the extreme C terminus of the dynein heavy chain control the rate-limiting step of product release in the dynein enzymatic cycle through long range allosteric mechanisms

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