Structure and mechanisms of the DsbB–DsbA disulfide bond generation machine

AbstractAll organisms possess specific cellular machinery that introduces disulfide bonds into proteins newly synthesized and transported out of the cytosol. In E. coli, the membrane-integrated DsbB protein cooperates with ubiquinone to generate a disulfide bond, which is transferred to DsbA, a periplasmic dithiol oxido-reductase that serves as the direct disulfide bond donor to proteins folding oxidatively in this compartment. Despite the extensive accumulation of knowledge on this oxidation system, molecular details of the DsbB reaction mechanisms had been controversial due partly to the lack of structural information until our recent determination of the crystal structure of a DsbA–DsbB–ubiquinone complex. In this review we discuss the structural and chemical nature of reaction intermediates in the DsbB catalysis and the illuminated molecular mechanisms that account for the de novo formation of a disulfide bond and its donation to DsbA. It is suggested that DsbB gains the ability to oxidize its specific substrate, DsbA, having very high redox potential, by undergoing a DsbA-induced rearrangement of cysteine residues. One of the DsbB cysteines that are now reduced then interacts with ubiquinone to form a charge transfer complex, leading to the regeneration of a disulfide at the DsbB active site, and the cycle can begin anew

Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling

Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream membrane protein biogenesis factor YidC2 (YqjG) in Bacillus subtilis, but not in Escherichia coli. In the absence of structures of Gram-positive bacterial ribosomes, a molecular basis for species-specific stalling has remained unclear. Here we present the structure of a Gram-positive B. subtilis MifM-stalled 70S ribosome at 3.5-3.9 angstrom, revealing a network of interactions between MifM and the ribosomal tunnel, which stabilize a non-productive conformation of the PTC that prevents aminoacyl-tRNA accommodation and thereby induces translational arrest. Complementary genetic analyses identify a single amino acid within ribosomal protein L22 that dictates the species specificity of the stalling event. Such insights expand our understanding of how the synergism between the ribosome and the nascent chain is utilized to modulate the translatome in a species-specific manner

Structural basis of Sec-independent membrane protein insertion by YidC

[プレスリリース]バイオサイエンス研究科膜分子複合機能学研究室の塚崎智也准教授らの研究グループが、タンパク質を細胞膜に組み込むメカニズムを解明しました（2014/04/17）Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively1, 2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG3, 4, 5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane

Nascentome Analysis Uncovers Futile Protein Synthesis in Escherichia coli

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a “nascentome.” We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms

ダイチョウキン ニ オケル トリプトファン オペロン ノ チョウセツ キコウ : trpR trpS イデンシ サンブツカン ノ ソウゴ サヨウ

京都大学0048新制・課程博士理学博士甲第1204号理博第244号新制||理||158(附属図書館)UT51-47-G8京都大学大学院理学研究科化学専攻(主査)教授 由良 隆, 教授 高浪 満, 教授 香月 裕彦学位規則第5条第1項該当Kyoto UniversityDFA

Biochemical Characterization of a Mutationally Altered Protein Translocase: Proton Motive Force Stimulation of the Initiation Phase of Translocation

Protein translocation across the Escherichia coli plasma membrane is facilitated by concerted actions of the SecYEG integral membrane complex and the SecA ATPase. A secY mutation (secY39) affects Arg357, an evolutionarily conserved and functionally important residue, and impairs the translocation function in vivo and in vitro. In this study, we used the “superactive” mutant forms of SecA, which suppress the SecY39 deficiency, to characterize the mutationally altered SecY39EG translocase. It was found that SecY39-mediated preprotein translocation exhibited absolute dependence on the proton motive force. The proton motive force-dependent step proved to lie before signal peptide cleavage. We suggest that the proton motive force assists in the initiation phase of protein translocation

Membrane Protein Degradation by FtsH Can Be Initiated from Either End

FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli. In this paper we investigated how membrane-embedded substrates are recognized by this enzyme. We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence. Subsequent proteolysis should involve dislocation of the substrates into the cytosol. We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail. This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain. These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction. Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously. These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH