Ensemble cryo-EM uncovers inchworm-like translocation of a viral IRES through the ribosome

Internal ribosome entry sites (IRESs) mediate cap-independent translation of viral mRNAs. Using electron cryo-microscopy of a single specimen, we present five ribosome structures formed with the Taura syndrome virus IRES and translocase eEF2*GTP bound with sordarin. The structures suggest a trajectory of IRES translocation, required for translation initiation, and provide an unprecedented view of eEF2 dynamics. The IRES rearranges from extended to bent to extended conformations. This inchworm-like movement is coupled with ribosomal inter-subunit rotation and 40S head swivel. eEF2, attached to the 60S subunit, slides along the rotating 40S subunit to enter the A site. Its diphthamide-bearing tip at domain IV separates the tRNA-mRNA-like pseudoknot I (PKI) of the IRES from the decoding center. This unlocks 40S domains, facilitating head swivel and biasing IRES translocation via hitherto-elusive intermediates with PKI captured between the A and P sites. The structures suggest missing links in our understanding of tRNA translocation

Huntingtin facilitates polycomb repressive complex 2

Huntington's disease (HD) is caused by expansion of the polymorphic polyglutamine segment in the huntingtin protein. Full-length huntingtin is thought to be a predominant HEAT repeat α-solenoid, implying a role as a facilitator of macromolecular complexes. Here we have investigated huntingtin's domain structure and potential intersection with epigenetic silencer polycomb repressive complex 2 (PRC2), suggested by shared embryonic deficiency phenotypes. Analysis of a set of full-length recombinant huntingtins, with different polyglutamine regions, demonstrated dramatic conformational flexibility, with an accessible hinge separating two large α-helical domains. Moreover, embryos lacking huntingtin exhibited impaired PRC2 regulation of Hox gene expression, trophoblast giant cell differentiation, paternal X chromosome inactivation and histone H3K27 tri-methylation, while full-length endogenous nuclear huntingtin in wild-type embryoid bodies (EBs) was associated with PRC2 subunits and was detected with trimethylated histone H3K27 at Hoxb9. Supporting a direct stimulatory role, full-length recombinant huntingtin significantly increased the histone H3K27 tri-methylase activity of reconstituted PRC2 in vitro, and structure–function analysis demonstrated that the polyglutamine region augmented full-length huntingtin PRC2 stimulation, both in HdhQ111 EBs and in vitro, with reconstituted PRC2. Knowledge of full-length huntingtin's α-helical organization and role as a facilitator of the multi-subunit PRC2 complex provides a novel starting point for studying PRC2 regulation, implicates this chromatin repressive complex in a neurodegenerative disorder and sets the stage for further study of huntingtin's molecular function and the impact of its modulatory polyglutamine region

Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins

The chloride channel (ClC) protein family comprises both chloride (Cl(-)) channels and chloride/proton (Cl(-)/H(+)) antiporters. In prokaryotes and eukaryotes, these proteins mediate the movement of Cl(-) ions across the membrane. In eukaryotes, ClC proteins play a role in the stabilization of membrane potential, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification. Moreover, mutations in the genes encoding ClC proteins can cause genetic disease in humans. In prokaryotes, the Cl(-)/H(+) antiporters, such as ClC-ec1 found in Escherichia coli promote proton expulsion in the extreme acid-resistance response common to enteric bacteria. To date, structural and functional studies of the prokaryotic protein have revealed unique structural features, including complicated transmembrane topology with 18 α-helices in each subunit and an anion-coordinating region in each subunit. Several different approaches such as X-ray crystallography, NMR, biochemical studies, and molecular dynamics simulations have been applied to the study of ClC proteins. Continued study of the unique structure and function of this diverse family of proteins has the potential to lead to the development of novel therapeutic targets for neuronal, renal, bone, and food-borne diseases

Biochemical and biophysical approaches to study the structure and function of the chloride channel (ClC) family of proteins

The chloride channel (ClC) protein family comprises both chloride (Cl-) channels and chloride/proton (Cl/H+) antiporters. In prokaryotes and eukaryotes, these proteins mediate the movement of Cl- ions across the membrane. In eukaryotes, ClC proteins play a role in the stabilization of membrane potential, epithelial ion transport, hippocampal neuroprotection, cardiac pacemaker activity and vesicular acidification. Moreover, mutations in the genes encoding ClC proteins can cause genetic disease in humans. In prokaryotes, the Cl/H+ antiporters, such as ClC-ec1 found in Escherichia coli promote proton expulsion in the extreme acid-resistance response common to enteric bacteria. To date, structural and functional studies of the prokaryotic protein have revealed unique structural features, including complicated transmembrane topology with 18 alpha-helices in each subunit and an anion-coordinating region in each subunit. Several different approaches such as X-ray crystallography, NMR, biochemical studies, and molecular dynamics simulations have been applied to the study of CIC proteins. Continued study of the unique structure and function of this diverse family of proteins has the potential to lead to the development of novel therapeutic targets for neuronal, renal, bone, and food-borne diseases. (C) 2016 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved

Superdex 200 SEC profile of detergent exchange.

<p>Purified ClC-rm1 in DDM was exchanged to (a) DM or (b) OG. In both cases, particles were monodisperse. (c) Micrograph of negatively stained ClC-rm1 in OG. Scale bar 20 nm. (d) Representative class averages of ClC-rm1 in OG.</p

Topology of eukaryotic ClC proteins.

<p>A schematic diagram of the ClC protein containing transmembrane catalytic domain and cytoplasmic regulatory domain. The topology of the transmembrane domain is based on the ClC-ec1 protein. The 18 α helices are labeled from A-R. The two halves of the subunits are shown in two different shades of gray. The regions and sequences that contribute to the chloride selectivity filter are marked with black arrows and the respective conserved sequences are indicated (<i>amino acids are abbreviated with their one-letter codes</i>, <i>/ (forward slash)</i>: <i>or</i>, <i>X</i>: <i>any amino acid)</i>. The two cytoplasmic cystathionine beta synthase (CBS) subdomains are shown in rounded rectangles (CBS1 and CBS2).</p

Micrographs of negatively stained ClC-rm1 in DDM.

<p>(a) DDM-solubilized ClC-rm1 particles are monodisperse. The size of some particles are different from the rest (highlighted by black arrowheads). Scale bar 20 nm. (b) Representative single-particle class averages.</p