Miki Wada

Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe

Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1alpha-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg(2+) is not required for GDP binding to eRF3c. Mg(2+) at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.

Structural insights into eRF3 and stop codon recognition by eRF1.

Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1s M domain contacts eRF3s GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1s stimulatory effect on eRF3s GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1s putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.

Omnipotent role of archaeal elongation factor 1 alpha (EF1α) in translational elongation and termination, and quality control of protein synthesis

The molecular mechanisms of translation termination and mRNA surveillance in archaea remain unclear. In eukaryotes, eRF3 and HBS1, which are homologous to the tRNA carrier GTPase EF1α, respectively bind eRF1 and Pelota to decipher stop codons or to facilitate mRNA surveillance. However, genome-wide searches of archaea have failed to detect any orthologs to both GTPases. Here, we report the crystal structure of aRF1 from an archaeon, Aeropyrum pernix, and present strong evidence that the authentic archaeal EF1α acts as a carrier GTPase for aRF1 and for aPelota. The binding interface residues between aRF1 and aEF1α predicted from aRF1·aEF1α·GTP ternary structure model were confirmed by in vivo functional assays. The aRF1/eRF1 structural domain with GGQ motif, which corresponds to the CCA arm of tRNA, contacts with all three structural domains of aEF1α showing striking tRNA mimicry of aRF1/eRF1 and its GTPase-mediated catalysis for stop codon decoding. The multiple binding capacity of archaeal EF1α explains the absence of GTPase orthologs for eRF3 and HBS1 in archaea species and suggests that universal molecular mechanisms underlie translational elongation and termination, and mRNA surveillance pathways.

Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin

In vertebrates, the iron exporter ferroportin releases Fe2+ from cells into plasma, thereby maintaining iron homeostasis. The transport activity of ferroportin is suppressed by the peptide hormone hepcidin, which exhibits upregulated expression in chronic inflammation, causing iron-restrictive anaemia. However, due to the lack of structural information about ferroportin, the mechanisms of its iron transport and hepcidin-mediated regulation remain largely elusive. Here we report the crystal structures of a putative bacterial homologue of ferroportin, BbFPN, in both the outward- and inward-facing states. Despite undetectable sequence similarity, BbFPN adopts the major facilitator superfamily fold. A comparison of the two structures reveals that BbFPN undergoes an intra-domain conformational rearrangement during the transport cycle. We identify a substrate metal-binding site, based on structural and mutational analyses. Furthermore, the BbFPN structures suggest that a predicted hepcidin-binding site of ferroportin is located within its central cavity. Thus, BbFPN may be a valuable structural model for iron homeostasis regulation by ferroportin.

Molecular Pathobiology and Antigenic Variation of Pneumocystis carinii

Publisher Summary The clinical importance of Pneumocystis carinii is intensified by the fact that anti- P. carinii drugs have major problems of efficacy and toxicity. With the isolation of nucleic acids from this organism, there have been marked advances in the molecular biology of P. carinii , which led to a resurgence of pathobiological and clinical studies. It is noted that P. carinii is the only fungus that uses a genetic system to switch cell surface determinants. The complexity of major surface glycoprotein (MSG) gene expression implies that antigenic polymorphism and variability play an important role in P. carinii pathobiology. Antigenic variation may contribute to the prevalence of P. carinii pneumonia (PCP) in AIDS patients with a low number of CD4+ T cells, as compared with other microbes. Antigenic variability is used to maintain an opportunistic infection in a single animal host, rather than a series of relapses, because of the moderate alterations in the MSG structures. A site-specific endonuclease(s) may be an important target for further investigation into the genetic control of MSG switching.

Mutations in the G-domain of Ski7 cause specific dysfunction in non-stop decay.

Ski7 functions as a cofactor in both normal mRNA turnover and non-stop mRNA decay (NSD) mRNA surveillance in budding yeast. The N-terminal region of Ski7 (Ski7N) interacts with the ski-complex and the exosome. The C-terminal region of Ski7 (Ski7C) binds guanine nucleotides and shares overall sequence and structural homology with the proteins of the translational GTPase superfamily, especially the tRNA/tRNA-mimic carrier protein subfamilies such as EF1α, eRF3, and Hbs1. Previous reports showed that Ski7N polypeptide functions adequately in vivo, while Ski7C, if any, only slightly. Furthermore, Ski7C does not exhibit GTP-hydrolysing activities under normal conditions. Therefore, the physiological and functional significance of the conserved Ski7C is unclear. Here, we report strong genetic evidence suggesting differential roles for Ski7N and Ski7C in normal and specific mRNA turnover pathways by creating/isolating mutations in both Ski7N and Ski7C conserved motifs using indicator yeast strains. We concluded that Ski7C participates in mRNA surveillance as a regulatory module competitively with the Hbs1/Dom34 complex. Our results provide insights into the molecular regulatory mechanisms underlying mRNA surveillance.

A genetic approach for analyzing the co-operative function of the tRNA mimicry complex, eRF1/eRF3, in translation termination on the ribosome

During termination of translation in eukaryotes, a GTP-binding protein, eRF3, functions within a complex with the tRNA-mimicking protein, eRF1, to decode stop codons. It remains unclear how the tRNA-mimicking protein co-operates with the GTPase and with the functional sites on the ribosome. In order to elucidate the molecular characteristics of tRNA-mimicking proteins involved in stop codon decoding, we have devised a heterologous genetic system in Saccharomyces cerevisiae. We found that eRF3 from Pneumocystis carinii (Pc-eRF3) did not complement depletion of S. cerevisiae eRF3. The strength of Pc-eRF3 binding to Sc-eRF1 depends on the GTP-binding domain, suggesting that defects of the GTPase switch in the heterologous complex causes the observed lethality. We isolated mutants of Pc-eRF3 and Sc-eRF1 that restore cell growth in the presence of Pc-eRF3 as the sole source of eRF3. Mapping of these mutations onto the latest 3D-complex structure revealed that they were located in the binding-interface region between eRF1 and eRF3, as well as in the ribosomal functional sites. Intriguingly, a novel functional site was revealed adjacent to the decoding site of eRF1, on the tip domain that mimics the tRNA anticodon loop. This novel domain likely participates in codon recognition, coupled with the GTPase function.

Crystal structures of the TRIC trimeric intracellular cation channel orthologues

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

Heterologous Expression of Pneumocystis carinii Genes in Fission Yeast

The major cell surface glycoproteins (MSG) of P. carinii play a vital role in pathobiology and host-parasite interaction. MSGs are encoded by a family of polymorphic genes [2] distributed among all of the 14-15 chromosomes, and are expressed from a unique expression site termed UCS [3]. We have shown that a UCS site i s telomeric and that antigenic variation may be generated by sitespecific or homologous recombination between UCS and polymorphic MSG repertoires [ l , 31. Here we show that the UCS sequence is active to initiate transcription and translation in the fission yeast, Shizosaccharomyces pombe. Three other promoters of P. carinii also stimulated heterologous expression in S. pombe. MATEFULS AND METHODS. The promoter activities of UCS and other P. carinii segments were examined in S. pombe strain JY333 (h leu1 ade6-M216; a kind gift of M. Yamamoto, Univ. Tokyo) using gene fusions to [acZ (P-galactosidase). The following DNAs containing the authentic promoter and the initiator codon were substituted for the nmrl promoter of the expression vector pRYP1: UCS (nucleotide positions numbered from translation start site, -395 +23), H+ ATPase promoter (Patp, -302 +23), folic acid synthesis promoter (Pfas, -433 4 7 ) . and thymidylate synthase promoter (Pts, -413 +23). Note that these constructs were designed to initiate translation from the initiator codon of the inserted segments. S. pombe cells were transformed with these plasmids by electroporation. and Leu+ transformants were measured for P-galactosidase activity according to the method of Ito et a!. (manuscript submitted for publication). RESULTS AND DISCUSSION. Recent study of molecular taxonomy has shown that P. carinii is much more closely related to fungi such as Sacchuromyces cerevisiae or S. pombe than to protozoa [l]. Based on this prediction, we tested whether known P. carinii promoters (Le., Patp. Pfas and Pts) are capable of initiating synthesis of P-galactosidase in yeast using gene fusions to a IacZ reporter. As shown in Table 1, Patp, Pts and Pfas segments enhanced P-galactosidase synthesis in S. pombe (in this order), while a control plasmid lacking a promoter segment failed to synthesize. The level of expression by Patp was one tenth of that of the nmrl (no message in thiamine) promoter which is widely used as an

Tight interaction of eEF2 in the presence of Stm1 on ribosome

The stress-related protein Stm1 interacts with ribosomes, and is implicated in repressing translation. Stm1 was previously studied both in vivo and in vitro by cell-free translation systems using crude yeast lysates, but its precise functional mechanism remains obscure. Using an in vitro reconstituted translation system, we now show that Stm1 severely inhibits translation through its N-terminal region, aa 1 to 107, and this inhibition is antagonized by eEF3. We found that Stm1 stabilizes eEF2 on the 80 S ribosome in the GTP-bound form, independently of eEF2s diphthamide modification, a conserved post-translational modification at the tip of domain IV. Systematic analyses of N- or C-terminal truncated mutants revealed that the core region of Stm1, aa 47 to 143, is crucial for its ribosome binding and eEF2 stabilization. Stm1 does not inhibit the 80 S-dependent GTPase activity of eEF2, at least during the first round of GTP-hydrolysis. The mechanism and the role of the stable association of eEF2 with the ribosome in the presence of Stm1 are discussed in relation to the translation repression by Stm1.