Importin-9 wraps around the H2A-H2B core to act as nuclear importer and histone chaperone.

We report the crystal structure of nuclear import receptor Importin-9 bound to its cargo, the histones H2A-H2B. Importin-9 wraps around the core, globular region of H2A-H2B to form an extensive interface. The nature of this interface coupled with quantitative analysis of deletion mutants of H2A-H2B suggests that the NLS-like sequences in the H2A-H2B tails play a minor role in import. Importin-9•H2A-H2B is reminiscent of interactions between histones and histone chaperones in that it precludes H2A-H2B interactions with DNA and H3-H4 as seen in the nucleosome. Like many histone chaperones, which prevent inappropriate non-nucleosomal interactions, Importin-9 also sequesters H2A-H2B from DNA. Importin-9 appears to act as a storage chaperone for H2A-H2B while escorting it to the nucleus. Surprisingly, RanGTP does not dissociate Importin-9•H2A-H2B but assembles into a RanGTP•Importin-9•H2A-H2B complex. The presence of Ran in the complex, however, modulates Imp9-H2A-H2B interactions to facilitate its dissociation by DNA and assembly into a nucleosome

Engineering the Controlled Assembly of Filamentous Injectisomes in E. coli K-12 for Protein Translocation into Mammalian Cells.

Bacterial pathogens containing type III protein secretion systems (T3SS) assemble large needle-like protein complexes in the bacterial envelope, called injectisomes, for translocation of protein effectors into host cells. The application of these molecular syringes for the injection of proteins into mammalian cells is hindered by their structural and genomic complexity, requiring multiple polypeptides encoded along with effectors in various transcriptional units (TUs) with intricate regulation. In this work, we have rationally designed the controlled expression of the filamentous injectisomes found in enteropathogenic Escherichia coli (EPEC) in the nonpathogenic strain E. coli K-12. All structural components of EPEC injectisomes, encoded in a genomic island called the locus of enterocyte effacement (LEE), were engineered in five TUs (eLEEs) excluding effectors, promoters and transcriptional regulators. These eLEEs were placed under the control of the IPTG-inducible promoter Ptac and integrated into specific chromosomal sites of E. coli K-12 using a marker-less strategy. The resulting strain, named synthetic injector E. coli (SIEC), assembles filamentous injectisomes similar to those in EPEC. SIEC injectisomes form pores in the host plasma membrane and are able to translocate T3-substrate proteins (e.g., translocated intimin receptor, Tir) into the cytoplasm of HeLa cells reproducing the phenotypes of intimate attachment and polymerization of actin-pedestals elicited by EPEC bacteria. Hence, SIEC strain allows the controlled expression of functional filamentous injectisomes for efficient translocation of proteins with T3S-signals into mammalian cells

Dimerization of VirD2 Binding Protein Is Essential for Agrobacterium Induced Tumor Formation in Plants

10.1371/journal.ppat.1003948PLoS Pathogens103

Structures of Tetrahymena’s respiratory chain reveal the diversity of eukaryotic core metabolism

Respiration is a core biological energy-converting process whose last steps are carried out by a chain of multisubunit complexes in the inner mitochondrial membrane. To probe the functional and structural diversity of eukaryotic respiration, we examined the respiratory chain of the ciliate Tetrahymena thermophila (Tt). Using cryo-electron microscopy on a mixed sample, we solved structures of a supercomplex between Tt complex I (Tt-CI) and Tt-CIII2 (Tt-SC I+III2) and a structure of Tt-CIV2. Tt-SC I+III2 (~2.3 megadaltons) is a curved assembly with structural and functional symmetry breaking. Tt-CIV2 is a ~2.7-megadalton dimer with more than 50 subunits per protomer, including mitochondrial carriers and a TIM83-TIM133-like domain. Our structural and functional study of the T. thermophila respiratory chain reveals divergence in key components of eukaryotic respiration, thereby expanding our understanding of core metabolism

The Mysterious Multitude: Structural Perspective on the Accessory Subunits of Respiratory Complex I

Complex I (CI) is the largest protein complex in the mitochondrial oxidative phosphorylation electron transport chain of the inner mitochondrial membrane and plays a key role in the transport of electrons from reduced substrates to molecular oxygen. CI is composed of 14 core subunits that are conserved across species and an increasing number of accessory subunits from bacteria to mammals. The fact that adding accessory subunits incurs costs of protein production and import suggests that these subunits play important physiological roles. Accordingly, knockout studies have demonstrated that accessory subunits are essential for CI assembly and function. Furthermore, clinical studies have shown that amino acid substitutions in accessory subunits lead to several debilitating and fatal CI deficiencies. Nevertheless, the specific roles of CI's accessory subunits have remained mysterious. In this review, we explore the possible roles of each of mammalian CI's 31 accessory subunits by integrating recent high-resolution CI structures with knockout, assembly, and clinical studies. Thus, we develop a framework of experimentally testable hypotheses for the function of the accessory subunits. We believe that this framework will provide inroads towards the complete understanding of mitochondrial CI physiology and help to develop strategies for the treatment of CI deficiencies

Resting mitochondrial complex I from Drosophila melanogaster adopts a helix-locked state

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway 'deactive' state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover

Structure of GrlR-GrlA complex that prevents GrlA activation of virulence genes

10.1038/ncomms3546Nature Communications4