Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry.

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules.

Structure of the γ-tubulin ring complex-capped microtubule.

Microtubules are composed of α-tubulin and β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.

Structure of the γ-tubulin ring complex-capped microtubule.

Unlabelled: Microtubules are composed of α/β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the filament's long-axis. We report that the human ∼2.

Targeting allostery in the Dynein motor domain with small molecule inhibitors.

Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S.

Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function.

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a "lumenal bridge" (LB).

MZT Proteins Form Multi-Faceted Structural Modules in the γ-Tubulin Ring Complex.

Microtubule organization depends on the γ-tubulin ring complex (γ-TuRC), a ∼2.3-MDa nucleation factor comprising an asymmetric assembly of γ-tubulin and GCP2-GCP6. However, it is currently unclear how the γ-TuRC-associated microproteins MZT1 and MZT2 contribute to the structure and regulation of the holocomplex.

Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex.

The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at ∼3.8 Å resolution, revealing an asymmetric, cone-shaped structure.

The CryoEM structure of the ribosome maturation factor Rea1.

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol.

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.

How dynein and dynactin transport cargos: a structural perspective.

Recent structures of the dynein motor in three different conformations reveal how it uses ATP hydrolysis to move along microtubules. Attention is now turning to how cytoplasmic dynein-1 and dynactin act together to carry cargos. Cryo-electron microscopy (cryo-EM) has revealed the structure of dynactin and how it binds dynein in the presence of a cargo adaptor protein Bicaudal-D2 (BICD2).

The structure of the dynactin complex and its interaction with dynein.

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.

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.

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.

The structure of sulfoindocarbocyanine 3 terminally attached to dsDNA via a long, flexible tether.

Fluorescence resonance energy transfer (FRET) is an important source of long-range distance information in macromolecules. However, extracting maximum information requires knowledge of fluorophore, donor and acceptor, positions on the macromolecule. We previously determined the structure of the indocarbocyanine fluorophores Cy3 and Cy5 attached to DNA via three-carbon atom tethers, showing that they stacked onto the end of the helix in a manner similar to an additional basepair.