Articles in 2012

In order to locate the voltage-dependent gate in the MthK potassium channel, intracellular quaternary ammonium blockers are used for electrophysiology and crystallographic analyses. The data conclusively show that the inactivation gate is located at the selectivity filter and not at the cytoplasmic bundle crossing entrance.

A biochemical approach is used to understand how the ULK1 complex integrates signals from ATG proteins during autophagy. The interaction between ULK1-associated protein FIP200 and ATG16L1 of the ATG5 complex is revealed. This interaction is important for autophagy induced by amino acid starvation but not by glucose deprivation.

The transcription machinery must locate specific promoter sequences among a vast excess of nonspecific DNA. Real-time single-molecule experiments with E. coli RNA polymerase, combined with theoretical calculations, suggest that facilitated diffusion does not contribute to promoter targeting at physiologically relevant protein concentrations but that instead the promoter search is dominated by three-dimensional diffusion.

Studies of the hepatitis C virus internal ribosome entry site (IRES) mechanism have focused on how IRES assembles an 80S ribosome at the start codon. Structural and functional analyses demonstrate that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause conformational changes and that the mutated domain decreases IRES activity by inhibiting ribosome translocation and, thereby, translation elongation.

The COPII cage, formed by Sec13 and Sec31, organizes other proteins into a lattice on the endoplasmic-reticulum membrane and is involved in transporting cargo from the endoplasmic reticulum to the Golgi apparatus. A combination of cryo-EM and H/D-exchange MS analyses leads to a 12-Å-resolution model of the COPII cage, yielding insight into its architecture and assembly.

A cell-based screen for intronic splicing silencers revealed ten sequence motifs that inhibited splicing in human cells and either enhanced or inhibited exon inclusion when inserted into exons. Identification of trans-acting splicing factors for each motif revealed a complex network, which suggests that cis elements function differently in distinct cellular contexts, depending on the regulatory factors present.

Dengue virus has two membrane proteins, E and M, which undergo dramatic structural changes during the life cycle of the virus. The 3.5-Å cryo-EM structure of the mature prefusion Dengue virion reveals the detailed interactions between E and M, providing insight into how conformational changes are triggered.

The transmembrane export apparatus regulates protein secretion through bacterial type III secretion systems. New structural data indicate that MxiA, a major component of the apparatus, assembles in a nonameric ring. This and additional structural information provide a framework for understanding how protein secretion is controlled.

Glucocorticoid receptor (GR) transactivates genes containing the response element GRE. GR can also mediate transrepression of genes by binding to the so-called negative GRE (nGRE). The interaction between GR and nGRE is now revealed by structural and functional approaches, showing that two GR monomers bind nGRE in a unique conformation and with strong negative cooperativity.

HLA-DM interacts with MHCII and promotes peptide exchange. This activity of HLA-DM is regulated by HLA-DO. The crystal structure of the HLA-DO–HLA-DM complex along with mutagenesis and kinetic analyses reveal that HLA-DO adopts a classical MHCII structure and competitively inhibits HLA-DM's activity on MHCII.

Formins regulate actin nucleation and filament elongation through their conserved FH2 domain. The formin FMNL3 induces assembly of filopodia, and now the crystal structure of its FH2 domain in complex with actin, together with functional analyses, provides insight into formin's activities.

The exoribonuclease Eri1 binds the stem-loop of histone mRNAs, but the functional significance of this interaction has been unclear. New studies now indicate that 3A oligouridylation of histone mRNAs enables the Lsm1–7 complex to bind the oligo(U) tail and to interact with Eri1, whose catalytic activity degrades the double-stranded stem-loop structure.

The ATG12~ATG5 conjugate promotes the transfer of the ubiquitin-like protein LC3 to phosphatidylethanolamine (PE), a modification required for autophagosome formation. Structural and biochemical analyses reveal the determinants for ATG12~ATG5 binding to ATG16 and the E3 ligase ATG3, and indicate how the conjugate stimulates PE–LC3 formation.

The eukaryotic ribosome-associated complex (RAC) chaperone is poorly understood. Structural analyses now provide insight into the catalytic inactivity and possible functions of the Ssz1 subunit and reveal that RAC interacts with the ribosome via the Zuo1 subunit. RAC crouches over the ribosomal exit tunnel, where its conformation may be controlled by the ribosomal expansion segment ES27.

Previous work has implied that the ATPase domain of ISWI chromatin remodelers cooperates with a DNA-binding accessory domain to achieve remodeling. Quantitative biochemical analyses now reveal that the ATPase domain exists in two conformations and that DNA binding induces the catalytically active conformation. The ATPase domain has an intrinsic ability to bind and remodel nucleosomes, which suggests that it acts autonomously.

Measles virus hemagglutinin (MVH) can bind to different cell surface receptors in the human host. CD46, the first identified MVH receptor, is used mainly by vaccine strains, whereas clinical strains can use SLAM on macrophages and dendritic cells and nectin-4 on epithelial cells. The crystal structure of MVH in complex with the outermost ectodomain of nectin-4 is now presented, revealing a potential target site for drug development.

Histone chaperone RbAp48 interacts with histones H3–H4 and delivers them to a second histone chaperone, ASF1, to be assembled into new nucleosomes. These interactions are now investigated, revealing that RbAp48 binds H3–H4 heterodimers (but not tetramers) and causes conformational changes in their core fold. Moreover, an allosteric mechanism facilitates exchange of H3–H4 between RbAp48 and ASF1.

Post-translational modifications are one way in which GTPase functions can be regulated. Monoubiquitination of Lys147 of Ras has been shown to promote tumorigenesis. New data now indicate that this modification promotes Ras activation by impairing GTP hydrolysis catalyzed by GTPase-activating proteins.

DnaK targets protein aggregates to ClpB. New data show that DnaK also activates ClpB in a species-specific manner through direct interactions with the M domain of ClpB, stabilizing a derepressed state that increases the ATP hydrolysis and protein disaggregation activities of the chaperone. Also in this issue, Oguchi et al. show how the M domain of ClpB acts as a reversible toggle to regulate these activities.

In the E. coli Hsp100/Hsp70 system, the M domain of ClpB is essential for ClpB cooperation with DnaK and DnaJ and for protein disaggregation, but its function was largely unknown. New biochemical and biophysical data now indicate that the M domain acts as a molecular toggle that reversibly interacts with the AAA-1 domain of ClpB to regulate ATP hydrolysis and protein disaggregation activities. Also in this issue, Seyffer et al. show how DnaK interactions with M domain further enhance ClpB activity.