Architecture of the yeast elongator complex

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub-complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two-lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator

Insights into the mode of action of a putative zinc transporter CzrB in thermus thermophilus

peer-reviewedThis paper was obtained through PEER (Publishing and the Ecology of European Research) http://www.peerproject.euThe crystal structures of the cytoplasmic domain of the putative zinc transporter CzrB in the apoand zinc-bound forms reported herein are consistent with the protein functioning in vivo as a homodimer. NMR, X-ray scattering and size exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modelling with the Zn2+ / H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of its zinc cargo. Since the cytoplasmic domain may exist in the cell as an independent, soluble protein a proposal is advanced that it functions as a metallochaperone and that it regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB

Architecture of the yeast elongator complex

The highly conserved eukaryotic Elongator complex performs specific chemical modifications on wobble base uridines of tRNAs, which are essential for proteome stability and homeostasis. The complex is formed by six individual subunits (Elp1-6) that are all equally important for its tRNA modification activity. However, its overall architecture and the detailed reaction mechanism remain elusive. Here, we report the structures of the fully assembled yeast Elongator and the Elp123 sub-complex solved by an integrative structure determination approach showing that two copies of the Elp1, Elp2, and Elp3 subunits form a two-lobed scaffold, which binds Elp456 asymmetrically. Our topological models are consistent with previous studies on individual subunits and further validated by complementary biochemical analyses. Our study provides a structural framework on how the tRNA modification activity is carried out by Elongator

Structures and activities of the Elongator complex and Its cofactors

Elongator is a highly conserved eukaryotic protein complex consisting of two sets of six Elp proteins, while homologues of its catalytic subunit Elp3 are found in all the kingdoms of life. Although it was originally described as a transcription elongation factor, cumulating evidence suggests that its primary function is catalyzing tRNA modifications. In humans, defects in Elongator subunits are associated with neurological disorders and cancer. Although further studies are still required, a clearer picture of the molecular mechanism of action of Elongator and its cofactors has started to emerge within recent years that have witnessed significant development in the field. In this review we summarize recent Elongator-related findings provided largely by crystal structures of several subunits of the complex, the electron microscopy structure of the entire yeast holoenzyme, as well as the structure of the Elongator cofactor complex Kti11/Kti13

Structure of the Elongator cofactor complex Kti11/Kti13 provides insight into the role of Kti13 in Elongator-dependent tRNA modification

Modification of wobble uridines of many eukaryotic tRNAs requires the Elongator complex, a highly conserved six-subunit eukaryotic protein assembly, as well as the Killer toxin-insensitive (Kti) proteins 11-14. Kti11 was additionally shown to be implicated in the biosynthesis of diphthamide, a post-translationally modified histidine of translation elongation factor 2. Recent data indicate that iron-bearing Kti11 functions as an electron donor to the [4Fe-4S] cluster of radical S-Adenosylmethionine enzymes, triggering the subsequent radical reaction. We show here that recombinant yeast Kti11 forms a stable 1 : 1 complex with Kti13. To obtain insights into the function of this heterodimer, the Kti11/Kti13 complex was purified to homogeneity, crystallized, and its structure determined at 1.45 A resolution. The importance of several residues mediating complex formation was confirmed by mutagenesis. Kti13 adopts a fold characteristic of RCC1-like proteins. The seven-bladed beta-propeller consists of a unique mixture of four- and three-stranded blades. In the complex, Kti13 orients Kti11 and restricts access to its electron-carrying iron atom, constraining the electron transfer capacity of Kti11. Based on these findings, we propose a role for Kti13, and discuss the possible functional implications of complex formation. DATABASE: Structural data have been submitted to the Protein Data Bank under accession number 4X33

Use of folding modulators to improve heterologous protein production in escherichia coli

Despite the fundamental importance of E. coli in the manufacture of a wide range of biotechnological and biomedical products, extensive process and/or target optimisation is routinely required in order to achieve functional yields in excess of low mg/l levels. Molecular chaperones and folding catalysts appear to present a panacea for problems of heterologous protein folding in the organism, due largely to their broad substrate range compared with, e. g., protein-specific mutagenesis approaches. Painstaking investigation of chaperone overproduction has, however, met with mixed - and largely unpredictable - results to date. The past 5 years have nevertheless seen an explosion in interest in exploiting the native folding modulators of E. coli, and particularly cocktails thereof, driven largely by the availability of plasmid systems that facilitate simultaneous, non-rational screening of multiple chaperones during recombinant protein expression. As interest in using E. coli to produce recombinant membrane proteins and even glycoproteins grows, approaches to reduce aggregation, delay host cell lysis and optimise expression of difficult-to-express recombinant proteins will become even more critical over the coming years. In this review, we critically evaluate the performance of molecular chaperones and folding catalysts native to E. coli in improving functional production of heterologous proteins in the bacterium and we discuss how they might best be exploited to provide increased amounts of correctly-folded, active protein for biochemical and biophysical studies

Molecular Aspects of Biogenesis of Escherichia coli Dr Fimbriae: Characterization of DraB-DraE Complexes

The Dr hemagglutinin of uropathogenic Escherichia coli is a fimbrial homopolymer of DraE subunits encoded by the dra operon. The dra operon includes the draB and draC genes, whose products exhibit homology to chaperone-usher proteins involved in the biogenesis of surface-located polymeric structures. DraB is one of the periplasmic proteins belonging to the superfamily of PapD-like chaperones. It possesses two conserved cysteine residues characteristic of the FGL subfamily of Caf1M-like chaperones. In this study we obtained evidence that DraB cysteines form a disulfide bond in a mature chaperone and have the crucial function of forming the DraB-DraE binary complex. Expression experiments showed that the DraB protein is indispensable in the folding of the DraE subunit to a form capable of polymerization. Accumulation of DraB-DraE(n) oligomers, composed of head-to-tail subunits and the chaperone DraB, was observed in the periplasm of a recombinant E. coli strain which expressed DraB and DraE (but not DraC). To investigate the donor strand exchange mechanism during the formation of DraE oligomers, we constructed a series of DraE N-terminal deletion mutants. Deletion of the first three N-terminal residues of a potential donor strand resulted in a DraE protein lacking an oligomerization function. In vitro data showed that the DraE disulfide bond was not needed to form a binary complex with the DraB chaperone but was essential in the polymerization process. Our data suggest that assembly of Dr fimbriae requires a chaperone-usher pathway and the donor strand exchange mechanism