Long range allosteric control of cytoplasmic dynein ATPase activity by the stalk and C-terminal domains.
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.
Höök
Peter
P
Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA.
Mikami
Atsushi
A
Shafer
Beth
B
Chait
Brian T
BT
Rosenfeld
Steven S
SS
Vallee
Richard B
RB
D000244:Adenosine Diphosphate / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000495:Allosteric Site; D017607:Aluminum Compounds / Q000494:pharmacology; D000818:Animals; D002852:Chromatography, Ion Exchange; D003593:Cytoplasm / Q000378:metabolism*; D004164:Diphosphonates / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004398:Dyneins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005459:Fluorides / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism; D008954:Models, Biological; D015233:Models, Statistical; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D012997:Solvents / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry / Q000494:pharmacology; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology
D017607:Aluminum Compounds; D004164:Diphosphonates; D011994:Recombinant Proteins; D012997:Solvents; C016514:imidodiphosphonic acid; D014638:Vanadates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D004398:Dyneins; D005459:Fluorides; C032311:aluminum fluoride