Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.

Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.

Publisher Correction: The cytoplasmic dynein transport machinery and its many cargoes.

In Figure 1c of the original article, ARP1 was incorrectly labelled as ARP11. The highlight for reference 37 was mistakenly placed under reference 36 and the highlight for reference 29 should have also referred to reference 16 (instead of 19). The HTML and PDF versions of the article have now been corrected.

The cytoplasmic dynein transport machinery and its many cargoes.

Cytoplasmic dynein 1 is an important microtubule-based motor in many eukaryotic cells. Dynein has critical roles both in interphase and during cell division. Here, we focus on interphase cargoes of dynein, which include membrane-bound organelles, RNAs, protein complexes and viruses. A central challenge in the field is to understand how a single motor can transport such a diverse array of cargoes and how this process is regulated. The molecular basis by which each cargo is linked to dynein and its cofactor dynactin has started to emerge. Of particular importance for this process is a set of coiled-coil proteins - activating adaptors - that both recruit dynein-dynactin to their cargoes and activate dynein motility.

Structure of dynein-dynactin on microtubules shows tandem adaptor binding.

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor1-3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour4-6. Different coiled-coil adaptors are linked to different cargos7,8, and some share motifs known to contact sites on dynein and dynactin4,9-13. There is limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here we develop a cryo-electron microscopy processing pipeline to solve the high-resolution structure of dynein-dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how they relate to motile behaviour. We found that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.

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

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.

CryoET shows cofilactin filaments inside the microtubule lumen.

Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identities of these objects and mechanisms for their accumulation have not been conclusively established. Here, we used cryogenic electron tomography of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase with the small molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, we observed cofilin dephosphorylation, an activating modification, in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNA interference knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen.

Directionality of dynein is controlled by the angle and length of its stalk.

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. Kinesin and myosin motor families have a subclass that moves towards the opposite end of the microtubule or actin filament with respect to the rest of the motor family1,2, whereas all dynein motors that have been studied so far exclusively move towards the minus end of the microtubule3. Guided by cryo-electron microscopy and molecular dynamics simulations, we sought to understand the mechanism that underpins the directionality of dynein by engineering a Saccharomyces cerevisiae dynein that is directed towards the plus end of the microtubule. Here, using single-molecule assays, we show that elongation or shortening of the coiled-coil stalk that connects the motor to the microtubule controls the helical directionality of dynein around microtubules. By changing the length and angle of the stalk, we successfully reversed the motility towards the plus end of the microtubule. These modifications act by altering the direction in which the dynein linker swings relative to the microtubule, rather than by reversing the asymmetric unbinding of the motor from the microtubule. Because the length and angle of the dynein stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the minus end of the microtubule.

Engineered disulfide reveals structural dynamics of locked SARS-CoV-2 spike.

The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct "S-R/x3" to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

Dynein swings into action.

Motor proteins, such as dynein, use chemical energy from ATP hydrolysis to move along the cytoskeleton. Roberts et al. (2009) now describe the arrangement of subdomains in the motor domain of dynein and propose a model for how these regions function together in force generation.

Cryo-EM shows how dynactin recruits two dyneins for faster movement.

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail-dynactin-BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein-dynactin transport machine.

Structure of human cytoplasmic dynein-2 primed for its power stroke.

Members of the dynein family, consisting of cytoplasmic and axonemal isoforms, are motors that move towards the minus ends of microtubules. Cytoplasmic dynein-1 (dynein-1) plays roles in mitosis and cellular cargo transport, and is implicated in viral infections and neurodegenerative diseases. Cytoplasmic dynein-2 (dynein-2) performs intraflagellar transport and is associated with human skeletal ciliopathies. Dyneins share a conserved motor domain that couples cycles of ATP hydrolysis with conformational changes to produce movement. Here we present the crystal structure of the human cytoplasmic dynein-2 motor bound to the ATP-hydrolysis transition state analogue ADP.vanadate. The structure reveals a closure of the motor's ring of six AAA+ domains (ATPases associated with various cellular activites: AAA1-AAA6). This induces a steric clash with the linker, the key element for the generation of movement, driving it into a conformation that is primed to produce force. Ring closure also changes the interface between the stalk and buttress coiled-coil extensions of the motor domain. This drives helix sliding in the stalk which causes the microtubule binding domain at its tip to release from the microtubule. Our structure answers the key questions of how ATP hydrolysis leads to linker remodelling and microtubule affinity regulation.

DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes.

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein's core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

In vitro reconstitution of a highly processive recombinant human dynein complex.

Cytoplasmic dynein is an approximately 1.4 MDa multi-protein complex that transports many cellular cargoes towards the minus ends of microtubules. Several in vitro studies of mammalian dynein have suggested that individual motors are not robustly processive, raising questions about how dynein-associated cargoes can move over long distances in cells. Here, we report the production of a fully recombinant human dynein complex from a single baculovirus in insect cells. Individual complexes very rarely show directional movement in vitro. However, addition of dynactin together with the N-terminal region of the cargo adaptor BICD2 (BICD2N) gives rise to unidirectional dynein movement over remarkably long distances. Single-molecule fluorescence microscopy provides evidence that BICD2N and dynactin stimulate processivity by regulating individual dynein complexes, rather than by promoting oligomerisation of the motor complex. Negative stain electron microscopy reveals the dynein-dynactin-BICD2N complex to be well ordered, with dynactin positioned approximately along the length of the dynein tail. Collectively, our results provide insight into a novel mechanism for coordinating cargo binding with long-distance motor movement.

Review: Structure and mechanism of the dynein motor ATPase.

Dyneins are multiprotein complexes that move cargo along microtubules in the minus end direction. The largest individual component of the dynein complex is the heavy chain. Its C-terminal 3500 amino-acid residues form the motor domain, which hydrolyses ATP in its ring of AAA+ (ATPases associated with diverse cellular activities) domains to generate the force for movement. The production of force is synchronized with cycles of microtubule binding and release, another important prerequisite for efficient motility along the microtubule. Although the large scale conformational changes that lead to force production and microtubule affinity regulation are well established, it has been largely enigmatic how ATP-hydrolysis in the AAA+ ring causes these rearrangements. The past five years have seen a surge of high resolution information on the dynein motor domain that finally allowed unprecedented insights into this important open question. This review, part of the "ATP and GTP hydrolysis in Biology" special issue, will summarize our current understanding of the dynein motor mechanism with a special emphasis on the recently obtained crystal and EM structures. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 557-567, 2016.

Crystal clear insights into how the dynein motor moves.

Dyneins are motor proteins that move along microtubules. They have many roles in the cell. They drive the beating of cilia and flagella, move cargos in the cytoplasm and function in the mitotic spindle. Dyneins are large and complex protein machines. Until recently, the way they move was poorly understood. In 2012, two high-resolution crystal structures of the >2500-amino-acid dynein motor domain were published. This Commentary will compare these structures and integrate the findings with other recent studies in order to suggest how dynein works. The dynein motor produces movement in a manner that is distinct from myosin and kinesin, the other cytoskeletal motors. Its powerstroke is produced by ATP-induced remodelling of a protein domain known as the linker. It binds to microtubules through a small domain at the tip of a long stalk. Dynein communicates with the microtubule-binding domain by an unconventional sliding movement of the helices in the stalk coiled-coil. Even the way the two motor domains in a dynein dimer walk processively along the microtubule is unusual.

A structural analysis of the AAA+ domains in Saccharomyces cerevisiae cytoplasmic dynein.

Dyneins are large protein complexes that act as microtubule based molecular motors. The dynein heavy chain contains a motor domain which is a member of the AAA+ protein family (ATPases Associated with diverse cellular Activities). Proteins of the AAA+ family show a diverse range of functionalities, but share a related core AAA+ domain, which often assembles into hexameric rings. Dynein is unusual because it has all six AAA+ domains linked together, in one long polypeptide. The dynein motor domain generates movement by coupling ATP driven conformational changes in the AAA+ ring to the swing of a motile element called the linker. Dynein binds to its microtubule track via a long antiparallel coiled-coil stalk that emanates from the AAA+ ring. Recently the first high resolution structures of the dynein motor domain were published. Here we provide a detailed structural analysis of the six AAA+ domains using our Saccharomycescerevisiae crystal structure. We describe how structural similarities in the dynein AAA+ domains suggest they share a common evolutionary origin. We analyse how the different AAA+ domains have diverged from each other. We discuss how this is related to the function of dynein as a motor protein and how the AAA+ domains of dynein compare to those of other AAA+ proteins.

Dynein and dynactin move long-range but are delivered separately to the axon tip.

Axonal transport is essential for neuronal survival. This is driven by microtubule motors including dynein, which transports cargo from the axon tip back to the cell body. This function requires its cofactor dynactin and regulators LIS1 and NDEL1. Due to difficulties imaging dynein at a single-molecule level, it is unclear how this motor and its regulators coordinate transport along the length of the axon. Here, we use a neuron-inducible human stem cell line (NGN2-OPTi-OX) to endogenously tag dynein components and visualize them at a near-single molecule regime. In the retrograde direction, we find that dynein and dynactin can move the entire length of the axon (>500 µm). Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with the initiation of dynein transport. Intriguingly, in the anterograde direction, dynein/LIS1 moves faster than dynactin/NDEL1, consistent with transport on different cargos. Therefore, neurons ensure efficient transport by holding dynein/dynactin on cargos over long distances but keeping them separate until required.

Molecular mechanism of dynein-dynactin complex assembly by LIS1.

Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

Stepping dynamics of dynein characterized by MINFLUX.

Cytoplasmic dynein is a dimeric motor that drives minus-end directed transport on microtubules (MTs). To couple ATP hydrolysis to a mechanical step, a dynein monomer must be released from the MT before undergoing a conformational change that generates a bias towards the minus end. However, the dynamics of dynein stepping have been poorly characterized by tracking flexible regions of the motor with limited resolution. Here, we developed a cysteine-light mutant of yeast dynein and site-specifically labeled its MT-binding domain in vitro. MINFLUX tracking at sub-millisecond resolution revealed that dynein hydrolyzes one ATP per step and takes multitudes of 8 nm steps at physiological ATP. Steps are preceded by the transient movement towards the plus end. We propose that these backward "dips" correspond to MT release and subsequent diffusion of the stepping monomer around its MT-bound partner before taking a minus-end-directed conformational change of its linker. Our results reveal the order of sub-millisecond events that result in a productive step of dynein.