Research Article

The structure of the dynactin complex and its interaction with dynein

See allHide authors and affiliations

Science  27 Mar 2015:
Vol. 347, Issue 6229, pp. 1441-1446
DOI: 10.1126/science.aaa4080

Making a molecular motor fit for purpose

Dynactin is an essential cofactor of the microtubule motor, cytoplasmic dynein. Dynactin contains 23 subunits built around a short filament of an actin-related protein (Arp1). How dynactin is assembled, how it functions with dynein, and why it is built around an actin-like filament is unclear. Urnavicius et al. combined cryo–electron microscopy structural studies and a crystal structure to determine the three-dimensional architecture of dynactin and how it interacts with dynein.

Science, this issue p. 1441


Dynactin is an essential cofactor for the microtubule motor cytoplasmic dynein-1. We report the structure of the 23-subunit dynactin complex by cryo-electron microscopy to 4.0 angstroms. Our reconstruction reveals how dynactin is built around a filament containing eight copies of the actin-related protein Arp1 and one of β-actin. The filament is capped at each end by distinct protein complexes, and its length is defined by elongated peptides that emerge from the α-helical shoulder domain. A further 8.2 angstrom structure of the complex between dynein, dynactin, and the motility-inducing cargo adaptor Bicaudal-D2 shows how the translational symmetry of the dynein tail matches that of the dynactin filament. The Bicaudal-D2 coiled coil runs between dynein and dynactin to stabilize the mutually dependent interactions between all three components.

Dynactin works with the cytoplasmic dynein-1 motor (dynein) to transport cargos along the microtubule cytoskeleton (13). Together, these protein complexes maintain the cell’s spatial organization, return components from the cell’s periphery, and assist with cellular division (4). Mutations in either complex cause neurodegeneration (5), and both can be co-opted by viruses that travel to the nucleus (6). Dynein and dynactin are similar in size and complexity. Dynein contains two copies of six different proteins and has a mass of 1.4 MD. Dynactin, at ~1.0 MD, contains more than 20 subunits, corresponding to 11 different proteins. Dynactin is built around a filament of actin-related protein 1 (Arp1). In analogy to actin, the filament has a barbed end and a pointed end, each capped by a different protein complex. On top sits the shoulder domain (7) from which emerges a long projection, corresponding to dynactin’s largest subunit, p150Glued (DCTN1) (8).

Despite the presence of a dynein binding site in p150Glued (911), purified dynein and dynactin form a stable complex only in the presence of the cargo adaptor Bicaudal-D2 (BICD2) (1214), a coiled-coil protein associated with transport of vesicles, mRNAs, and nuclei (15). This interaction activates dynein and converts it into a highly processive motor (13, 14).

Current models for dynactin’s architecture (7, 16, 17) and its interaction with dynein (13, 14) come from low-resolution negative-stain and platinum-shadowing electron microscopy (EM). A number of questions remain. What makes the filament in dynactin short and defined, when purified Arp1 filaments vary in length (18)? How does dynein bind to dynactin, and why does the interaction require BICD2 (1214)? Why does dynactin, the cofactor for a microtubule motor, contain an actin-related filament? To address these questions, we took advantage of recent advances in cryo-electron microscopy (cryo-EM) (19) to improve our structural understanding of dynactin.

Dynactin structure determination

Dynactin is a challenging target for cryo-EM (17). This complex’s extreme preferred orientation on EM grids makes it hard to obtain the broad distribution of views required for a three-dimensional (3D) reconstruction. Furthermore, dynactin’s thin elongated shape limits its contrast, making it difficult to assign views accurately. We overcame these hurdles (20) to determine cryo-EM maps of native dynactin purified from pig brain (fig. S1), initially at 6.3 Å (Dynactin-1 in table S1) and subsequently at 4.0 Å overall and 3.5 Å in the dynactin filament (Dynactin-2, -3, and -4 in table S1; Fig. 1, A and B; fig. S2; and movies S1 and S2). We used both maps to build a model of dynactin (Fig. 1C and table S2). The filament and pointed-end–capping protein Arp11 were built de novo and refined (table S3). Homology models of the barbed-end–capping protein CapZαβ (21) and the pointed-end proteins p25 (DCTN5) and p27 (DCTN6) (22) were fitted into density. The pointed-end protein p62 and the shoulder—which contains p150Glued (DCTN1), p50 (dynamitin or DCTN2), and p24 (DCTN3)—were built as backbone models (Fig. 1D).

Fig. 1 Cryo-EM structure of dynactin.

(A) A 4.0 Å cryo-EM map of dynactin segmented and colored according to its components. (B) A density map of a β strand and an adenosine diphosphate (ADP) molecule in Arp1-C. (C) A molecular model of dynactin. (D) A 6.3 Å cryo-EM map showing helices in the dynactin shoulder.

The dynactin filament contains eight Arp-1 subunits and one β-actin

The dynactin filament is nine subunits long and consists of two protofilaments that wrap around each other (Fig. 1, A and C): five subunits (A, C, E, G, and I) in the top protofilament and four (B, D, F, and H) in the bottom. The presence of β-actin in the filament is controversial (7, 23). Our cryo-EM map was of sufficient quality (fig. S3A) to show that subunit H is β-actin (β-actin–H), whereas the others are Arp1 (Arp1-A to Arp1-I). We confirmed the presence of Arp1 and β-actin at an 8:1 ratio by mass spectrometry (MS)–based label-free quantitative proteomic analysis (table S4).

Capping the dynactin filament

The dynactin filament is similar to that of actin (24), consistent with the high (53%) sequence identity between β-actin and Arp1 (fig. S3B). Both consist of four subdomains surrounding a nucleotide binding site (fig. S4). A key contact within the filament is the subdomain-2 loop binding the groove between subdomains 1 and 3 on the neighboring subunit. Blocking this interaction provides a mechanism to cap both actin and dynactin filaments.

At the barbed end of dynactin, a CapZαβ heterodimer binds across both protofilaments. The C-terminal helices (tentacles) of CapZα and CapZβ fit into the groove between subdomains 1 and 3 on Arp1-B and Arp1-A (Fig. 2A, fig. S5, and movie S3) and prevent further subunit binding. CapZαβ interacts with dynactin in the same way as proposed for the actin filament (25). However, there is a loop (called the “plug”) (24) that contains four negatively charged residues in Arp1 but only one in actin (Fig. 2B). This loop is close to a cluster of four positively charged residues on CapZα, suggesting that CapZαβ binds Arp1 with a higher affinity than actin. This explains why a pool of CapZαβ remains bound to dynactin but not to actin, when most CapZαβ is depleted by small interfering RNA (26). The tight binding of CapZαβ reflects its role in stabilizing dynactin’s structure.

Fig. 2 Capping the dynactin filament.

(A) The barbed end is capped by CapZαβ. (B) CapZαβ contains five positive residues (blue) that interact with four negative residues (orange) on Arp1. The equivalent loop in actin contains only one negative residue. C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; I, Ile; K, Lys; L, Leu; M, Met; R, Arg; S, Ser. (C) The short Arp11 subdomain-2 loop prevents further subunit addition to the bottom protofilament. (D) Arp11 caps the top protofilament by binding the subdomain-2 loop of Arp1-I and sterically blocking (asterisk) subsequent subunit binding. (E) The pointed-end complex: p62 extends over Arp11 to touch β-actin–H. (F) p25 and p27 pack end-on to Arp11 as a continuation of the bottom protofilament.

At the pointed end, the bottom protofilament ends in β-actin, whereas the top ends in Arp1 (Fig. 1A). This creates a distinctive binding site for Arp11, the most evolutionarily distant of all the actin-related proteins (27). Our structural data reveal how a single Arp11 subunit can cap both protofilaments. The bottom protofilament directly binds Arp11, preventing further subunit addition because its subdomain-2 loop is too short (27) (Fig. 2C). Subunit addition to the Arp1-I subunit on the top protofilament is blocked sterically by subdomain 4 of Arp11 (asterisk in Fig. 2D) and also because the Arp1-I subdomain-2 loop is sequestered by Arp11 (Fig. 2D).

Arp11 binds p25, p27, and p62 to form the pointed-end complex (Fig. 2E). The p25 and p27 subunits consist of a triangular β-sheet structure (22) followed by an α helix (fig. S6). They pack side to edge (fig. S6A) and bind end-on to Arp11 (Fig. 2F). The interaction is reinforced by p62, which wraps around the Arp11:p25-p27 contact site (Fig. 2E). Only Arp11 directly caps the pointed end, suggesting that the other components have a different role, such as cargo attachment (28), and explaining why some fungal species contain Arp11 but lack p25, p27, and p62 (29).

The p150Glued projection extends more than 50 nm from the shoulder

Previous antibody labeling showed that p150Glued forms dynactin’s shoulder projection (7). Existing models suggest that it is 24 nm long and contains the p150Glued N-terminal Cap-Gly domain and the CC1A coiled coil (Fig. 3A) (7, 8). Owing to its flexibility, the projection is not visible in our high-resolution EM maps. However, we determined an 8.6 Å structure from a subset of particles (Fig. 3B, table S1, and fig. S7) in which it is visible because it docks against the side of dynactin (fig. S8, A and B). The projection is more than 50 nm long and contains three coiled coils, which we assigned to those in p150Glued on the basis of their length (fig. S8C). An ~18-nm coiled coil (CC2) emerges from the shoulder and joins a globular domain consisting of a dimer (fig. S8D) of two ~40-kD subunits [intercoiled domain (ICD)]. Another ~24-nm coiled coil (CC1B) extends from the ICD before doubling back for ~18 nm (CC1A). Our model predicts that the Cap-Gly domain at the N terminus of CC1A is located close to the ICD. This is not visible in our structure, owing to its flexibility and because the majority of our dynactin contains the shorter isoform of p150Glued (p135), which lacks a Cap-Gly domain (Fig. 3A). Our 50-nm projection is similar in appearance to structures observed in the images of dynactin viewed by rotary shadowing (7). Furthermore, the interaction made by CC1A folding back onto CC1B agrees with recent biochemical data (30).

Fig. 3 The architecture of the p150Glued projection and shoulder.

(A) Schematic models of p150Glued. (B) An 8.6 Å cryo-EM structure with a docked p150Glued projection, colored according to (A). (C) Native MS of dynactin reveals the mass of the intact complex. m/z, mass/charge ratio. (D) Tandem MS confirms the subunit composition of the complex. (E) The shoulder contains two arms (red and blue) that emerge from a dimerization center (green) and end in hook and paddle domains. (F) The C terminus of the p150Glued dimer enters the shoulder and splits into separate helices.

The shoulder’s symmetry is broken by binding the dynactin filament

Previous models suggest that the shoulder consists mainly of p150Glued (8). Our finding that most of p150Glued is in the projection implies that the shoulder contains predominantly p24 and p50. We confirmed that dynactin contains four copies of p50 and two copies of p24 (27), because the mass of dynactin calculated from this stoichiometry matches (table S5) that measured by MS (1,066,889 daltons) (Fig. 3C). We verified this composition by tandem MS (Fig. 3D) and by measuring the resulting subcomplexes (table S5).

The shoulder’s flexibility results in a lower-resolution cryo-EM map and makes it challenging to assign individual α helices to specific proteins. Instead, the structures reveal an intrinsic twofold symmetry of the shoulder that was not obvious in previous images of dynactin (17). The shoulder contains two identical arms (Fig. 3, E and F) made of bundles of three α helices (Fig. 1D) that meet at a dimerization domain (fig. S9A). The end of each arm meets another short bundle of helices at an acute angle (hook domain) and a variably positioned paddle domain (Fig. 3E and fig. S9B). The symmetry between the two arms is broken as they twist to contact the dynactin filament.

The stoichiometry of p24 and p50 and their predicted helical and coiled-coil structures (8, 31) suggest that each arm contains one helix from p24 and two from p50. The length of p24 (186 residues) is similar to the length of one arm, whereas that of p50 (401 residues) is longer, suggesting that p50 contributes to other structures such as the hook or paddle domain. The two p150Glued copies enter the shoulder between the two arms (Fig. 3F) and then split and run along each arm before joining the hook domains. Thus, the role of the p150Glued C terminus is to stick the two p50-p24 arms together.

Extended peptides from the shoulder span the length of the dynactin filament

The invariant size of the dynactin filament implies that some mechanism specifies its length (7, 17, 32). The shoulder is the best candidate for dynactin’s molecular ruler. Its main body contacts four Arp1 subunits close to the barbed end (fig. S10). In addition, four extended regions (ERs) emerge from the shoulder and coat the rest of the filament (Fig. 4). The ends of all four are structurally identical (fig. S11, A to D), which suggests that they correspond to p50, the only tetramer in dynactin (8). The ERs are most likely the N termini of p50, which are predicted to be unstructured, contain a sequence that fits the clearest parts of the ER density (fig. S11E), and are able to displace the shoulder from the dynactin filament (26). Two pairs of ERs emerge from each shoulder paddle (Fig. 4, A and B). One of each pair contacts the top protofilament: ER-1 runs from Arp1-C to Arp1-E and ER-2 from Arp1-G to Arp1-I (Fig. 4C). The other reaches down to contact the bottom protofilament: ER-3 runs from Arp1-B to Arp1-D, and ER-4 contacts Arp1-F (Fig. 4D). The ends of all four ERs occupy a positively charged groove on the dynactin filament that is equivalent to the tropomyosin binding groove on actin (33) (fig. S12, A and B). Although it is structurally different from tropomyosin (24), the N terminus of p50 is similarly rich in negatively charged residues (fig. S12C), which explains why both bind an equivalent site.

Fig. 4 Shoulder peptides coat and measure the dynactin filament.

(A and B) Four extended regions (ER1 to ER4) connect to the shoulder paddles. (C) ER1 and ER2 cover the full length of the top Arp1 protofilament. (D) ER3 and ER4 cover the bottom protofilament subunits (Arp1-B, -D, and -F) but not the β-actin–H subunit.

The shoulder and its ERs contact every filament subunit except for β-actin–H (fig. S12, D and E), suggesting the following model for dynactin assembly. The shoulder and ERs recruit eight Arp1s and stabilize their polymerization into a structure with five subunits on the top protofilament and three on the bottom. The gap in position H is filled by β-actin, perhaps owing to actin’s high abundance in the cell. The interface formed by Arp1-I and β-actin–H specifically recruits Arp11. Together with CapZαβ binding to the barbed end, this results in a highly stable complex of an exactly defined length.

Dynein and BICD2 bind the dynactin filament

Dynein, dynactin, and the N terminus of BICD2 (BICD2N) form a stable complex only when all three components are present (1214). In this dynein-dynactin-BICD2N (DDB) complex, dynein binds dynactin via its tail, whereas its motor domains remain flexible (13). Currently there are no 3D structures of either the dynein tail or its interaction with dynactin. We therefore formed a stable tail-dynactin-BICD2N (TDB) complex (fig. S13) and determined its structure by cryo-EM to 8.2 Å (fig. S14 and movie S4).

The dynein tail binds directly to the Arp1 filament (Fig. 5, A and B), stretching from β-actin–H to the barbed end. The interaction is stabilized by a ~270-residue coiled coil of BICD2N that runs the length of the filament. Projections of the TDB complex are very similar to negative-stain images of the DDB complex (fig. S15) (13), suggesting that the flexible, C-terminal motor domains of dynein lie close to the barbed end of dynactin (fig. S15). To determine the orientation of BICD2N, we removed the N-terminal green fluorescent protein tag (table S1 and fig. S16) and showed that the globular density at one end disappeared (fig. S16). Therefore, the BICD2N N terminus lies close to the barbed end, and its C terminus emerges from the pointed end. The C terminus of BICD2 and the pointed-end complex of dynactin, which are implicated in cargo binding (28, 34), are thus diametrically opposed to the dynein motor domains (Fig. 5A).

Fig. 5 The dynein tail and its interaction with dynactin and BICD2N.

(A) Cartoon model of the dynein tail–dynactin–BICD2N complex (TDB). (B) An 8.2 Å cryo-EM structure of TDB. (C) An N-terminal domain dimerizes the dynein heavy chain (DHC) elongated domains, which wrap around the dynein intermediate chain (DIC2). (D) Crystal structure of the S. cerevisiae DHC N terminus (Dyn11-557). (E) The Dyn11-557 structure fits well into the cryo-EM map. (F) The translational symmetry of DHC chains 1 and 2 matches the dynactin filament. (G) Interaction of chain 1 with BICD2N and dynactin (asterisks). (H) The second interaction site of chain 1 with dynactin is solely mediated by BICD2N.

The dynein heavy chain contains an N-terminal dimerization domain

The dynein tail consists of two copies of the dynein heavy chain (DHC), intermediate chain (DIC2), light intermediate chain (DLIC1), and light chains (Roadblock, Tctex, and LC8). The tail, within the TDB complex, contains two elongated S-shaped domains corresponding to the DHCs: chain 1 and chain 2 (Fig. 5C). The DHC C termini, which contain the binding site for DLIC1 (35), are mainly disordered (Fig. 5B). The middle of each DHC wraps around a circular density corresponding in size, shape, and position (35) to the WD40 β propeller of DIC2 (Fig. 5C and fig. S17). Toward their N termini, the two DHC chains are joined by a small (~40 kDa) globular domain (Fig. 5C). We hypothesized that this domain represents a previously unknown dimerization domain of the DHC itself. To verify this, we determined a crystal structure of the N-terminal 557 amino acids of the Saccharomyces cerevisiae DHC (Dyn11-557) to 5 Å resolution (Fig. 5D, fig. S18, and table S6). This shows two elongated domains, made up of bundles of α helices, that fit well with the helices observed by cryo-EM (fig. S18, C and D). Furthermore, it reveals that the elongated domains are joined by an N-terminal dimerization domain (Fig. 5D).

In the crystal structure, the elongated domains are related by rotational symmetry, with their C termini pointing in opposite directions (Fig. 5D). In the TDB structure, each elongated domain has rotated about the flexible connection to the dimerization domain (movie S5) so that they lie parallel to each other (Fig. 5E). This is probably caused by the light-chain–mediated dimerization of DIC2 (36, 37), which serves to hold the DHCs together toward their C termini.

The symmetry in the dynein tail matches the dynactin filament

The N termini of the elongated domains have translational symmetry (a sideways movement relates one onto the other) (Fig. 5E), which matches that of their binding sites on the filament (Fig. 5F). They bind adjacent clefts between Arp1-D and -F (chain 1) and Arp1-F and β-actin–H (chain 2) (Fig. 5, F and G). These sites are equivalent to the myosin motor binding site on actin (fig. S19 and movie S6). The translational symmetry between DHC chains is lost toward their C termini as chain 2 twists relative to chain 1 (Fig. 5C). Chain 2 makes no further contacts with the dynactin filament, whereas chain 1 binds it again at Arp1-A and -C (Fig. 5H).

BICD2N is involved in all interactions between the dynein tail and the dynactin filament (Fig. 5, F, G, and H; and movie S7). In the clefts between Arp1-D and -F and between Arp1-F and β-actin–H, BICD2N stabilizes the interaction of the DHC chains with the filament (Fig. 5G). At the Arp1-A and -C site, BICD2N sits between chain 1 and dynactin. The network of contacts from BICD2N to dynein, BICD2N to dynactin, and dynein to dynactin explains why all three components must be present to form a stable complex (1214). The long BICD2N coiled coil may be suited as a cargo adaptor because it spans the length of the dynactin filament. Many other dynein adaptors—including TRAK1 and -2 (38); RILP (39); and Rab11-Fip3, Hook3, and Spindly (14)—contain coiled coils and may recruit dynein and dynactin in a similar way. The requirement to form a three-way complex would reduce the chance of stochastic binding of dynein to its cargos.

The shoulder coats three sides of the filament (fig. S12, D and E), leaving the front face free for interaction with BICD2N and the dynein tail. We do not observe a direct interaction between the shoulder and dynein in our structure. However, the well-reported interaction between the p150Glued CC1 region of dynactin and the N terminus of DIC2 (11) is too flexible to be visualized directly. The p150Glued projection binds to the same face of the dynactin filament as BICD2N (Figs. 3B and 5, A and B), and both occupy the same cleft on the pointed-end complex (fig. S8B and Fig. 5B). Thus, BICD2N binding could free p150Glued CC1 to make contact with DIC2 and so add an additional contact that stabilizes the TDB complex.

How does recruitment of dynactin by a cargo adaptor (13, 14) activate dynein? Both the microtubule binding Cap-Gly (14) and DIC2 binding CC1B (30) regions of p150Glued have been implicated. Our structure raises a third, but not mutually exclusive, possibility. Studies with artificially dimerized dynein motor domains suggest that they self-associate in an auto-inhibited conformation unless they are sufficiently separated (40). We suggest that dynactin activates the motor domains by reorienting the two DHCs. Both DHC N termini are anchored parallel to each other, but the C termini are forced to twist apart because only one chain binds the second site on dynactin. This hypothesis explains why dynactin is built around an actin-like filament. The translational symmetry of the filament matches that of the DHC N termini, whereas the filament length provides additional binding sites that force dynein to adopt its active conformation.

Supplementary Materials

Materials and Methods

Figs. S1 to S19

Tables S1 to S6

References (4165)

Movies S1 to S7

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We thank S. Scheres and X. Bai for cryo-EM advice; G. McMullan, C. Savva, J. Grimmett, and T. Darling for technical support; S. Leech for fresh pig brains; V. Beilsten-Edmands for assistance with proteomic analyses; S. Bullock, R. McKenney, and J. Pennell for comments on the manuscript; and Y. Toyoshima for sharing her unpublished negative-stain EM model of the p150Glued projection, which helped us to interpret our observations. This work was funded by the Medical Research Council, UK (MC_UP_A025_1011), and a Wellcome Trust New Investigator Award (WT100387). Cryo-EM maps are deposited with the Electron Microscopy Data Bank (EMD-2854, EMD-2855, EMD-2856, EMD-2857, EMD-2860, EMD-2861, and EMD-2862), and coordinates are deposited with the Protein Data Bank (5AFT, 5AFU, and 5AFR). Author contributions: L.U. prepared dynactin and determined the TDB structure. K.Z. determined the structure of dynactin. A.G.D. and M.Y. determined the crystal structure of the DHC N terminus. C.M. and M.A.S. prepared the dynein tail complex. N.A.P. and C.V.R performed MS. A.P.C. initiated the project and designed the experiments.
View Abstract

Stay Connected to Science

Navigate This Article