Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains.
J Biol Chem
; 280(38): 33045-54, 2005 Sep 23.
Article
em En
| MEDLINE
| ID: mdl-16030013
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dineínas
/
Citoplasma
Tipo de estudo:
Risk_factors_studies
Limite:
Animals
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2005
Tipo de documento:
Article
País de afiliação:
Estados Unidos