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/dynactin to distinct cargos. Here we use electron microscopy (EM) and single molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased toward recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two. We find that the shift toward a double dynein complex increases both force and speed. A 3.5 Å cryo-EM 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 how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein/dynactin transport machine.

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/dynactin to distinct cargos. Here we use electron microscopy (EM) and single molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased toward recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two. We find that the shift toward a double dynein complex increases both force and speed. A 3.5 Å cryo-EM 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 how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein/dynactin transport machine.

The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F 1 domain. The bacterial F 1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b′ subunits, down a noncatalytic interface of the F 1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F 1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30°to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.Paracoccus denitrificans | ATP synthase | structure | regulation | proton translocation T he ATP synthases (F-ATPases) found in eubacteria, chloroplasts, and mitochondria are multiprotein molecular machines with a rotary action that provide most cellular ATP. Our understanding of how they work has come mainly from single-molecule studies of rotation made almost entirely on bacterial F-ATPases (1) and from structures of their constituent domains determined predominantly with enzymes from mitochondria (2-5). The most complete high-resolution structure contains about 85% of the bovine F-ATPase, built up from substructures determined by X-ray crystallography (2), within the constraints of an overall structure determined by cryo-EM (6). This model provides many details about the catalytic mechanism of the F 1 domain (2, 5); its mode of inhibition by the natural inhibitor protein of F 1 -ATPase, IF 1 (2, 4); and the design of the rotor, an ensemble of a membrane-bound ring of eight c-subunits, and the elongated central stalk, which penetrates into the catalytic F 1 domain. However, it lacks a crucial region that would help to explain how the enzyme uses the transmembrane proton-motive force produced by respiration or photosynthesis to generate the turning of the rotor in its membrane domain and other features that keep the proton-motive force coupled to the synthesis of ATP.Few structural studies have been carried out on the F-ATPases from eubacteria. Their subunit compositions are simpler than the subu...

The ζ subunit is a novel natural inhibitor of the α-proteobacterial F1FO-ATPase described originally in Paracoccus denitrificans. To characterize the mechanism by which this subunit inhibits the F1FO nanomotor, the ζ subunit of Paracoccus denitrificans (Pd-ζ) was analyzed by the combination of kinetic, biochemical, bioinformatic, proteomic, and structural approaches. The ζ subunit causes full inhibition of the sulfite-activated PdF1-ATPase with an apparent IC50 of 270 nM by a mechanism independent of the ε subunit. The inhibitory region of the ζ subunit resides in the first 14 N-terminal residues of the protein, which protrude from the 4-α-helix bundle structure of the isolated ζ subunit, as resolved by NMR. Cross-linking experiments show that the ζ subunit interacts with rotor (γ) and stator (α, β) subunits of the F1-ATPase, indicating that the ζ subunit hinders rotation of the central stalk. In addition, a putatively regulatory nucleotide-binding site was found in the ζ subunit by isothermal titration calorimetry. Together, the data show that the ζ subunit controls the rotation of F1FO-ATPase by a mechanism reminiscent of, but different from, those described for mitochondrial IF1 and bacterial ε subunits where the 4-α-helix bundle of ζ seems to work as an anchoring domain that orients the N-terminal inhibitory domain to hinder rotation of the central stalk.