The structure of the dynactin complex and its interaction with dynein

Abstract

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 4.0Å. Our reconstruction reveals how dynactin is built around a filament containing eight copies of the actin related protein Arp1 and one of β-actin. Capped at each end by distinct protein complexes, the length of the filament is defined by elongated peptides that emerge from the α-helical shoulder domain. A further 8.2Å 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 (1-3). They 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 6 different proteins and has a mass of 1.4 MDa. Dynactin, at about 1.0 MDa, contains more than 20 subunits, corresponding to 12 different proteins. Dynactin is built around a filament of actin related protein 1 (Arp1). In analogy to actin, the filament has a barbed 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 p150 Glued (DCTN1) (8).Despite the presence of a dynein binding site in p150 Glued (9-11), purified dynein and dynactin only form a stable complex in the presence of the cargo adaptor Bicaudal D2 † To whom correspondence should be addressed: cartera@mrc-lmb.cam.ac.uk. 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 DHC N-terminus crystal structure. C.M. and M.A.S. prepared the dynein tail complex. N.A.P. and C.V.R performed mass spectrometry. A.P.C. initiated the project and designed the experiments.

Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

“…The major regulator of dynein that is required for most of its cellular functions is dynactin, a ~ 1.2 MDa protein complex that consists of various copies of eleven different protein subunits (Karki & Holzbaur, 1999; Schroer, 2004; Urnavicius et al , 2015). Despite being the key dynein regulator, dynactin itself interacts only weakly with dynein (McKenney et al , 2014; Schlager et al , 2014).…”

“…Additional adaptor proteins that recruit dynein to cargoes stabilise this interaction, leading to ternary complex formation and activation of processive dynein motility (McKenney et al , 2014; Schlager et al , 2014). Ternary complex formation is thought to release dynein from its autoinhibited state, possibly by separating the two motor domains (Chowdhury et al , 2015; Urnavicius et al , 2015; Carter et al , 2016). …”

“…One example is the metazoan‐specific adapter protein Bicaudal‐D2 (BicD2) that is critical for bidirectional transport of mRNA particles and contributes to the positioning of the endoplasmic reticulum, the Golgi apparatus and the nucleus (Bullock & Ish‐Horowicz, 2001; Hoogenraad et al , 2001; Hoogenraad & Akhmanova, 2016). The N‐terminal coiled‐coil of BicD2 mediates the interaction between the dynein tail and dynactin, which is crucial for processive minus‐end‐directed dynein motion (McKenney et al , 2014; Schlager et al , 2014; Chowdhury et al , 2015; Urnavicius et al , 2015), whereas the C‐terminal part of BicD2 interacts with Rab receptors on cargo membranes (Hoogenraad et al , 2001; Hoogenraad & Akhmanova, 2016). …”

“…The critical dynactin subunit for plus‐end tracking is p150 Glued (called p150 here). Its N‐terminal CAP‐Gly domain protrudes from the shoulder of the dynactin complex (Chowdhury et al , 2015; Urnavicius et al , 2015) and binds directly to microtubules (Culver‐Hanlon et al , 2006; Peris et al , 1998; Ross et al , 2006), as well as to EBs (Honnappa et al , 2006). In vitro reconstitution experiments with a p150 fragment showed that the p150 CAP‐Gly domain and the first coiled‐coil of p150, which interacts with the intermediate chain of the dynein complex (Karki & Holzbaur, 1995; Vaughan & Vallee, 1995; King et al , 2003), were sufficient for mediating EB‐dependent end tracking of the human dynein complex (Duellberg et al , 2014).…”

“…The regulation of the balance between dynein microtubule end tracking and processive motility was, however, not studied in these experiments as this requires the presence of the entire dynactin complex. Furthermore, in a recent cryo‐electron microscopy structure of the dynactin complex, both the p150 CAP‐Gly domain and the first coiled‐coil appear buried in a groove of the dynactin shoulder, raising the question of whether p150 in the context of the entire dynactin complex can mediate EB‐dependent dynein end tracking at all, or if additional factors might be needed to release a potential autoinhibition of dynactin (Urnavicius et al , 2015). …”