Cryo-EM structure of the inactive ribosome complex accumulated in chick embryo cells in cold-stress conditions.

Here, we present the high-resolution structure of the Gallus gallus 80S ribosome obtained from cold-treated chicken embryos. The translationally inactive ribosome complex contains elongation factor eEF2 with GDP, SERPINE1 mRNA binding protein 1 (SERBP1) and deacylated tRNA in the P/E position, showing common features with complexes already described in mammals. Modeling of most expansion segments of G. gallus 28S ribosomal RNA allowed us to identify specific features in their structural organization and to describe areas where a marked difference between mammalian and avian ribosomes could shed light on the evolution of the expansion segments. This study provides the first structure of an avian ribosome, establishing a model for future structural and functional studies on the translational machinery in Aves.

Purification and characterization of native human elongation factor 2.

Human elongation factor 2 is the translocase that is responsible for the movement of tRNA from the A- to P- and P- to E-site on the ribosome during the elongation phase of translation. Being a vital factor of protein biosynthesis, its function is highly controlled and regulated. It has been implicated in numerous diseases and pathologies, and as such it is important to have a source for isolated pure and active protein for biomedical and biochemical studies. Here we report development of a purification protocol for native human elongation factor 2 from HEK-293S cells. The resulting protein is active, pure, has an intact diphtamide and is obtainable in yields suitable for functional and structural studies.

Structural analysis of ribosomal RACK1 and its role in translational control.

Receptor for Activated C-Kinase 1 (RACK1) belongs to the WD40 family of proteins, known to act as scaffolding proteins in interaction networks. Accordingly, RACK1 is found to have numerous interacting partners ranging from kinases and signaling proteins to membrane bound receptors and ion channels. Interestingly, RACK1 has also been identified as a ribosomal protein present in all eukaryotic ribosomes. Structures of eukaryotic ribosomes have shown RACK1 to be located at the back of the head of the small ribosomal subunit. This suggests that RACK1 could act as a ribosomal scaffolding protein recruiting regulators of translation to the ribosome, and several studies have in fact found RACK1 to play a role in regulation of translation. To fully understand the role of RACK1 we need to understand whether the many reported interaction partners of RACK1 bind to free or ribosomal RACK1. In this review we provide a structural analysis of ribosome-bound RACK1 to provide a basis for answering this fundamental question. Our analysis shows that RACK1 is tightly bound to the ribosome through highly conserved and specific interactions confirming RACK1 as an integral ribosomal protein. Furthermore, we have analyzed whether reported binding sites for RACK1 interacting partners with a proposed role in translational control are accessible on ribosomal RACK1. Our analysis shows that most of the interaction partners with putative regulatory functions have binding sites that are available on ribosomal RACK1, supporting the role of RACK1 as a ribosomal signaling hub. We also discuss the possible role for RACK1 in recruitment of ribosomes to focal adhesion sites and regulation of local translation during cell spreading and migration.

Structure of TSA2 reveals novel features of the active-site loop of peroxiredoxins.

Saccharomyces cerevisiae TSA2 belongs to the family of typical 2-Cys peroxiredoxins, a ubiquitously expressed family of redox-active enzymes that utilize a conserved peroxidatic cysteine to reduce peroxides. Typical 2-Cys peroxiredoxins have been shown to be involved in protection against oxidative stress and in hydrogen peroxide signalling. Furthermore, several 2-Cys peroxiredoxins, including S. cerevisiae TSA1 and TSA2, are able to switch to chaperone activity upon hyperoxidation of their peroxidatic cysteine. This makes the sensitivity to hyperoxidation of the peroxidatic cysteine a very important determinant for the cellular function of a peroxiredoxin under different cellular conditions. Typical 2-Cys peroxiredoxins exist as dimers, and in the course of the reaction the peroxidatic cysteine forms a disulfide with a resolving cysteine located in the C-terminus of its dimeric partner. This requires a local unfolding of the active site and the C-terminus. The balance between the fully folded and locally unfolded conformations is of key importance for the reactivity and sensitivity to hyperoxidation of the different peroxiredoxins. Here, the structure of a C48S mutant of TSA2 from S. cerevisiae that mimics the reduced state of the peroxidatic cysteine has been determined. The structure reveals a novel conformation for the strictly conserved Pro41, which is likely to affect the delicate balance between the fully folded and locally unfolded conformations of the active site, and therefore the reactivity and the sensitivity to hyperoxidation. Furthermore, the structure also explains the observed difference in the pKa values of the peroxidatic cysteines of S. cerevisiae TSA1 and TSA2 despite their very high sequence identity.