Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins

Drosophila selenophosphate synthetase 1 regulates vitamin B6 metabolism: prediction and confirmation

<p>Abstract</p> <p>Background</p> <p>There are two selenophosphate synthetases (SPSs) in higher eukaryotes, SPS1 and SPS2. Of these two isotypes, only SPS2 catalyzes selenophosphate synthesis. Although SPS1 does not contain selenophosphate synthesis activity, it was found to be essential for cell growth and embryogenesis in <it>Drosophila</it>. The function of SPS1, however, has not been elucidated.</p> <p>Results</p> <p>Differentially expressed genes in <it>Drosophila </it>SL2 cells were identified using two-way analysis of variance methods and clustered according to their temporal expression pattern. Gene ontology analysis was performed against differentially expressed genes and gene ontology terms related to vitamin B6 biosynthesis were found to be significantly affected at the early stage at which megamitochondria were not formed (day 3) after <it>SPS1 </it>knockdown. Interestingly, genes related to defense and amino acid metabolism were affected at a later stage (day 5) following knockdown. Levels of pyridoxal phosphate, an active form of vitamin B6, were decreased by <it>SPS1 </it>knockdown. Treatment of SL2 cells with an inhibitor of pyridoxal phosphate synthesis resulted in both a similar pattern of expression as that found by <it>SPS1 </it>knockdown and the formation of megamitochondria, the major phenotypic change observed by <it>SPS1 </it>knockdown.</p> <p>Conclusions</p> <p>These results indicate that SPS1 regulates vitamin B6 synthesis, which in turn impacts various cellular systems such as amino acid metabolism, defense and other important metabolic activities.</p

Structural mimicry between SLA/LP and Rickettsia surface antigens as a driver of autoimmune hepatitis: insights from an in silico study

Background: Autoimmune hepatitis (AIH) is a chronic, progressive liver disease, characterized by continuing hepatocellular inflammation and necrosis. A subgroup of AIH patients presents specific autoantibodies to soluble liver antigen/liver-pancreas (SLA/LP) protein, which is regarded as a highly specific diagnostic marker. Autoantigenic SLA/LP peptides are targeted by CD4(+) T cells, and restricted by the allele HLA-DRB1*03:01, which confers disease susceptibility in Europeans and Americans. A positively charged residue at position 71 has been indicated as critical for AIH susceptibility in all of the HLA alleles identified to date. Though the exact molecular mechanisms underlying pathogenesis of AIH are not clear, molecular mimicry between SLA/LP and viral/bacterial antigens has been invoked. Methods: The immunodominant region of SLA/LP was used as query in databank searches to identify statistically significant similarities with viral/bacterial peptides. Homology modeling and docking was used to investigate the potential interaction of HLA-DRB1*03:01 with the identified peptides. By molecular mechanics means, the interactions and energy of binding at the HLA binding site was also scrutinized. Results: A statistically significant structural similarity between the immunodominant regions of SLA/LP and a region of the surface antigen PS 120 from Rickettsia spp. has been detected. The interaction of the SLA/LP autoepitope and the corresponding Rickettsia sequence with the allele HLA-DRB1*03:01 has been simulated. The obtained results predict for both peptides a similar binding mode and affinity to HLA-DRB1*03:01. A "hot spot" of interaction between HLA-DRB1*03:01 and PS 120 is located at the P4 binding pocket, and is represented by a salt bridge involving Lys at position 71 of the HLA protein, and Glu 795 of PS120 peptide. Conclusions: These findings strongly support the notion that a molecular mimicry mechanism can trigger AIH onset. CD4(+) T cells recognizing peptides of SLA/LP could indeed cross-react with foreign Rickettsia spp. antigens. Finally, the same analysis suggests a molecular explanation for the importance of position 71 in conferring the susceptibility of the allele HLA-DRB1*03:01 to AIH. The lack of a positive charge at such position could prevent HLA alleles from binding the foreign peptides and triggering the molecular mimicry event