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.

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s(-1). The disulfide bond-mediated conformation switch results in a metastable form that is locally strained by approximately 3 kcal mol(-1). On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.

Mitochondrial dysfunctions activate retrograde signaling from mitochondria to the nucleus. To identify transcription factors and their associated pathways that underlie mitochondrial retrograde signaling, we performed gene expression profiling of the cells engineered to have varying amounts of mitochondrial DNA with an A3243G mutation (mt3243) in the leucine transfer RNA (tRNA(Leu)), which reduces the abundance of proteins involved in oxidative phosphorylation that are encoded by the mitochondrial genome. The cells with the mutation exhibited reduced mitochondrial function, including compromised oxidative phosphorylation, which would activate diverse mitochondrial retrograde signaling pathways. By analyzing the gene expression profiles in cells with the mutant tRNA(Leu) and the transcription factors that recognize the differentially regulated genes, we identified 72 transcription factors that were potentially involved in mitochondrial retrograde signaling. We experimentally validated that the mt3243 mutation induced a retrograde signaling pathway involving RXRA (retinoid X receptor α), reactive oxygen species, kinase JNK (c-JUN N-terminal kinase), and transcriptional coactivator PGC1α (peroxisome proliferator-activated receptor γ, coactivator 1 α). This RXR pathway contributed to the decrease in mRNA abundances of oxidative phosphorylation enzymes encoded in the nuclear genome, thereby aggravating the dysfunction in oxidative phosphorylation caused by the reduced abundance of mitochondria-encoded enzymes of oxidative phosphorylation. Thus, matching transcription factors to differentially regulated gene expression profiles was an effective approach to understand mitochondrial retrograde signaling pathways and their roles in mitochondrial dysfunction.

The structure of MtrA, an essential gene product for the human pathogen Mycobacterium tuberculosis, has been solved to a resolution of 2.1 A. MtrA is a member of the OmpR/PhoB family of response regulators and represents the fourth family member for which a structure of the protein in its inactive state has been determined. As is true for all OmpR/PhoB family members, MtrA possesses an N-terminal regulatory domain and a C-terminal winged helix-turn-helix DNA-binding domain, with phosphorylation of the regulatory domain modulating the activity of the protein. In the inactive form of MtrA, these two domains form an extensive interface that is composed of the alpha4-beta5-alpha5 face of the regulatory domain and the C-terminal end of the positioning helix, the trans-activation loop, and the recognition helix of the DNA-binding domain. This domain orientation suggests a mechanism of mutual inhibition by the two domains. Activation of MtrA would require a disruption of this interface to allow the alpha4-beta5-alpha5 face of the regulatory domain to form the intermolecule interactions that are associated with the active state and to allow the recognition helix to interact with DNA. Furthermore, the interface appears to stabilize the inactive conformation of MtrA, potentially reducing the rate of phosphorylation of the N-terminal domain. This combination of effects may form a switch, regulating the activity of MtrA. The domain orientation exhibited by MtrA also provides a rationale for the variation in linker length that is observed within the OmpR/PhoB family of response regulators.