Microtubule Binding Domain from Mus musculus Cytoplasmic Dynein as a Fusion Protein with Seryl-tRNA Synthetase from Thermus thermophilus (PDB ID: 3ERR)
Created by: Erica Basque
There are 3 families of motor proteins known in the body, including kinesins, myosins, and dyneins. Of these motor proteins, the least is known about dynein as it is the only family of motor proteins in which the binding domain is separated from the catalytic domain by a 10 to 15 nm coiled-coil stalk (2). Because of this, it is hypothesized that kinesin and myosin evolved from a common ancestral protein while dynein emerged separately as a part of the AAA+ ATPase superfamily (9). The dynein family consists of two subunits, axonemal and cytoplasmic, the first of which is associated with movement of cilia and flagella and the latter of which is involved in a wide variety of functions throughout the cell including transport for vesicles, viruses, and mRNA as well as assisting in regulation of the mitotic checkpoint and organization of the golgi apparatus (4,5). A commonly referenced function of dynein is its movement of cargo along neuronal axons from the plus end to the minus end—that is, from the synapse to the cell body (5). As such, dysfunctions in dynein have been linked to diseases such as lissencephaly, male infertility, and neural degeneration among others (2). It is uncertain how information is passed along the coiled-coil stalk from the microtubule binding domain (MTBD) to the catalytic domain, and thus dynein MTBD has, in fairly recent years, become a topic of study. Formation of a fusion protein of MTBD from Mus musculus cytoplasmic dynein and seryl-tRNA synthetase (SRS) from Thermus thermophilus (PDB ID = 3ERR) allows for the stable crystallization of the MTBD and study of possible mechanisms of information propagation along the 15nm coiled coil stalk (2,4). The fusion protein’s molecular weight is 60,875.8 Da and its isoelectric point (pI) is 6.11 (6).
Dynein’s motor domain structure is a heavy chain which consists of an ATPase region of 6 AAA+ domains organized in a ring at one end, a “linker”, a 15 nm coiled-coil stalk, and a microtubule binding domain at the opposing end of the stalk (2). In the ring of AAA+ domains, only AAA1 through AAA4 bind nucleotides, including ATP at AAA1, while AAA5 and AAA6 are non-nucleotide binding. The first α-helix of the coiled coil stalk (CC1) originates from AAA4 and connects to the globular MTBD, which consists of 6 α-helices (H1-H6). The second coil of the stalk (CC2) returns from the MTBD to the AAA5 domain (2). Dynein also contains a non-motor region of coiled-coils which allow the protein to dimerize as well as associate with various light and intermediate chains to carry a wide variety of cargo. This area of dynein has a varying amino acid sequence depending upon the specific function of that dynein, which dictates the specific cargo of the dynein (9). (For a complete view of both heavy and light chains of dynein, refer to Image 1.) The fusion protein crystallizes only a portion of the coiled-coil and the MTBD while replacing the ATPase region with Thermus thermophilius SRS (2).
In intact cytoplasmic dynein, the binding and subsequent hydrolyzation of ATP in the catalytic site of the AAA+ ring induces conformation changes throughout the ring which are responsible for driving the movement of dynein down its microtubule (4). These conformational changes of the AAA+ ring cause a position change of the linker domain, which is thought to be the main force for motility. However, changes in the AAA+ ring also affect the binding affinity of the MTBD, allowing for the motion to actually occur. Studies performed with the MTBD and SRS fusion protein support the notion that it is conformational change in the coiled-coils which allow the information of ATP binding to travel between the catalytic domain and the MTBD (4).
Heptad repeats of 7 amino acids are a primary feature of coiled coils, including those found in dynein, and each heptad generally contains at least two hydrophobic amino acids (4). These hydrophobic amino acids interact and stack with each other, but in a somewhat unpredictable manner. The way in which the hydrophobic residues of one α-helix stack with the hydrophobic residues of another are referred to as registries and it is these registries which were altered in the fusion of dynein MTBD with SRS (4). Differing lengths of the coiled coil of the MTBD were fused with the coiled coils of SRS, resulting in two registries, referred to as a +β registry and α registry, which arise with a 4 residue heptad slip (8). The longer +β registry results in a low affinity microtubule biding state of the MTBD which would generally be associated with ATP biding in the AAA+ ring (4). The shorter α registry results in a high affinity microtubule binding state which would correlate to ATP hydrolysis and subsequent dissociation from the catalytic site as ADP and Pi (4). When the coiled-coil is observed in a shortened form, such as in the fusion protein, it prefers to adopt the +β registry (8). However, when the full length coiled coil and MTBD of dynein is observed in isolation, the α registry is favored, indicating that energetics are altered when the entire coiled coil is able to fold together (8).
An explanation for the different affinities of the two heptad registries may involve the way in which the two α-helices of the coiled coil are asymmetrically attached to the MTBD below two kinks formed by two highly conserved prolines (P3409 and P3285). In the MTBD, only H1, H3, and H6 are directly involved in the binding of microtubules (2). CC1 associates only with H1 and H3 while CC2 connects to all four of the other α-helices of the MTBD (2,4). Due to such a connection scheme CC2 remains stationary while CC1 is free to slide against CC2 during conformational changes. This leads to the movement of H1 and H3, both of which are involved in the binding of microtubules, thus providing an explanation for the changes in affinity with changes in registry (4). The binding of the MTBD to a microtubule may cause an additional conformational change which returns the coiled coil to the +β registry and propagate a signal back up to the AAA+ ATPase to hydrolyze the bound ATP (2).
The actual movement of dynein down the microtubule is facilitated by the linker domain on the AAA+ region. Binding of ATP to AAA1, in addition to changing MTBD to a low affinity conformation, also alters the conformation of the linker domain. In response to ATP binding, the AAA1 domain closes, propagating that conformational change to AAA2 and then subdomain 3 of the linker, which folds in a flexible region between subdomains 2 and 3 (3). This is a primed state of the dynein in which the protein is dissociated from the microtubule and the linker is prepared to perform a power stroke (Image 2). The hydrolysis of ATP and release of product ADP sends the linker back to its original conformation, producing a power stroke which moves the entire protein and its cargo forward (3), similar to the manner in which swinging ones legs forward on swing propels one in that direction. The release of the ATP ligand also returns the MTBD to its high affinity binding state.
A final important aspect of the movement of dynein on microtubules is its cooperation with kinesin, which moves in the opposing direction in the same area of the microtubule as dynein. Both motors are unidirectional, which raises the question of how each returns to the opposite end of the microtubule after transport of their cargo. It is suggested that at each end of the microtubule, known as the “turnaround zone”, the two complimentary motor proteins become active or inactive respectively and are carried back (10). In dynein’s case, after reaching the minus end of a microtubule it becomes inactive. This dynein would have been carrying an inactive kinesin, which activates at the minus end and carries the dynein back to the plus end along with its cargo (10) (Image 3).
However, there are multiple dynein and kinesin motors on each microtubule, begging the question of how they are both able to move along the same area of the microtubule simultaneously. Although dynein and kinesin evolved separately, they have converged to shared a binding site on the microtubule (9). When the two motors encounter each other along a section of microtubule, it is believed that dynein temporarily moves aside to allow kinesin to pass. This hypothesis arose because kinesin binds with higher affinity to the microtubule and there is also evidence for limited lateral movement of dynein (9).
Dynein performs a multitude of important functions in the body and as such is present in almost all animals and some plants. However, as dynein is unique among motor proteins, there is limited data on dynein from a large number of organisms. A Dali server analysis for similar 3D structure as well as a BLAST sequence analysis for primary structure similarity both returned the cytoplasmic dynein heavy chain of the amoeba Dictyostelium discoideum (PDB ID = 3VKH) as a homolog to Mus musculus dynein. Dali analysis was performed using the fusion protein and demonstrates the conservation of the 3D structure of the MTBD and coiled coil of dynein among organisms. BLAST analysis was performed using the full heavy chain residue sequence and shows that even between organisms as seemingly different as mice and amoeba, over 50% of the amino acid sequence is retained (69% positives).
Dynein is a unique motor protein which has only begun to truly be understood in approximately the last decade. It is a highly conserved AAA+ ATPase which facilitates movement along microtubules in a novel fashion rarely seen elsewhere. The fusion protein of mouse MTBD and bacterial SRS has allowed for observation of these movement mechanisms and the conformational changes which facilitate them. With understanding of these mechanisms comes the possibility of research into dysfunctions of dynein which lead to disease and possible solutions to such dysfunctions.