ABSTRACT
Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the "linker" domain, involving a hinge near its middle. Analysis of a mutant in which the linker "undocks" from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.
Subject(s)
Adenosine Triphosphate/chemistry , Axonemal Dyneins/chemistry , Chlamydomonas reinhardtii/enzymology , Dictyostelium/enzymology , Adenosine Diphosphate/chemistry , Axonemal Dyneins/ultrastructure , Axoneme/ultrastructure , Cryoelectron Microscopy , Microscopy, Video , Microtubules/chemistry , Microtubules/ultrastructure , Models, Molecular , Plant Proteins/chemistry , Plant Proteins/ultrastructure , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , Protozoan Proteins/chemistry , Protozoan Proteins/ultrastructure , Structural Homology, ProteinABSTRACT
We examined the functional roles of C-sequence, a 47-kDa non-AAA+ module at the C-terminal end of the 380-kDa Dictyostelium dynein motor domain. When the distal segment of the C-sequence was deleted from the motor domain, the single-molecule processivity of the dimerized motor domain was selectively impaired without its ensemble motile ability and ATPase activity being severely affected. When the hinge-like sequence between the distal and proximal C-sequence segments was made more or less flexible, the dimeric motor showed lower or higher processivity, respectively. These results suggest a potential function of the distal C-sequence segment as a modulator of processivity.
Subject(s)
Adenosine Triphosphate/metabolism , Cytoplasmic Dyneins/metabolism , Dictyostelium/metabolism , Microtubules/metabolism , Amino Acid Sequence , Binding Sites , Cytoplasmic Dyneins/chemistry , Cytoplasmic Dyneins/genetics , Dictyostelium/genetics , Molecular Sequence Data , Protein Multimerization , Sequence Homology, Amino AcidABSTRACT
Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by approximately 17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dynein's structure and mechanism.
Subject(s)
Dictyostelium/ultrastructure , Dyneins/metabolism , Protozoan Proteins/metabolism , Animals , Dictyostelium/metabolism , Dyneins/ultrastructure , Green Fluorescent Proteins/metabolism , Microscopy, Electron , Protozoan Proteins/ultrastructureABSTRACT
Dynein is an AAA+ (ATPase associated with various cellular activities)-type motor complex that utilizes ATP hydrolysis to actively drive microtubule sliding. The dynein heavy chain (molecular mass >500 kDa) contains six tandemly linked AAA+ modules and exhibits full motor activities. Detailed molecular dissection of this motor with unique architecture was hampered by the lack of an expression system for the recombinant heavy chain, as a result of its large size. However, the recent success of recombinant protein expression with full motor activities has provided a method for advances in structure-function studies in order to elucidate the molecular mechanism of force generation.
