Cryo‐EM reveals the complex architecture of dynactin's shoulder region and pointed end

Abstract

Dynactin is a 1.1 MDa complex that activates the molecular motor dynein for ultra‐processive transport along microtubules. In order to do this, it forms a tripartite complex with dynein and a coiled‐coil adaptor. Dynactin consists of an actin‐related filament whose length is defined by its flexible shoulder domain. Despite previous cryo‐EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high‐resolution information in these regions. Here we combine multiple cryo‐EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high‐resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder securely houses the p150 subunit and positions the four identical p50 subunits in different conformations to bind dynactin’s filament. The pointed end map allows us to build the first structure of p62 and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.

Conformational transitions of the mitotic adaptor Spindly underlie its interaction with Dynein and Dynactin

“…We then determined the crystal structure of human Spindly 1-100 (Table S1; only residues 2-97 were clearly resolved in the electron density), a fragment containing both the CC1 box and the CC1 SCM (Figure 1D-F). The structure demonstrated that Spindly forms a parallel dimeric coiled-coil, similar in its outline to that observed in structures of other adaptors captured in complex with DD (Lau et al, 2021; Urnavicius et al, 2018; Urnavicius et al, 2015). Thus, both Spindly FL and an N-terminal segment of Spindly exist as stable dimers.…”

“…We next attempted to investigate what forces contribute to maintain Spindly’s autoinhibited state. The pointed end (PE) subcomplex of Dynactin, which interacts with the conserved Spindly motif, has previously been shown to bind adaptors with limited but measurable binding affinity (Gama et al, 2017; Lau et al, 2021; Yeh et al, 2012). Thus, we developed a minimal recombinant adaptor-binding subcomplex of Dynactin, containing only the subunits of the pointed end-capping complex, p25, p27, p62 and Arp11 (Figure 3A).…”

“…After showing that Spindly WT does not interact with DD, we were eager to test whether the open Spindly mutants formed a super-complex with DD. For these assays, we were able to set up the production of recombinant human Dynactin, a significant technical challenge (Lau et al, 2021; Urnavicius et al, 2015). We produced Dynactin in the HEK293 expi expression system, using a modified version of the pBiG2 plasmid from the biGBac system (Weissmann et al, 2016) (Figure 6 – Supplement 1A).…”

“…The motility of Dynein requires the 1.1 MDa complex Dynactin (Carter et al, 2016; Reck-Peterson et al, 2018). Dynactin consists of 23 polypeptides and 11 individual subunits, organized in four main structural domains: 1) a central actin-like filament consisting of eight ARP1 subunits and one actin; 2) a four-subunit pointed-end capping complex including the subunits p25, p27, p62, Arp11; 3) a barbed-end capping complex containing CapZαβ; and 4) a shoulder domain containing p24, p150 glued , and p50/dynamitin (Chowdhury et al, 2015; Lau et al, 2021; Urnavicius et al, 2015).…”

Sequential dynein effectors regulate axonal autophagosome motility in a maturation-dependent pathway

“…6 ) and robust dynein–dynactin complex motility in vitro ( Schroeder and Vale, 2016 ). A popular hypothesis suggests the pointed-end complex protein p25 contains a mutually exclusive binding site that either binds a Spindly motif or the tail of autoinhibited p150; therefore, activator binding to p25 releases p150 from autoinhibition ( Cianfrocco et al, 2015 ; Schroeder and Vale, 2016 ; Qiu et al, 2018 ; Lau et al, 2021 ).…”