Crystallographic and NMR Evidence for Flexibility in Oligosaccharyltransferases and Its Catalytic Significance

Oligosaccharyltransferase (OST) catalyzes the transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. In the bacterium Campylobacter jejuni, a single-subunit membrane protein, PglB, catalyzes N-glycosylation. We report the 2.8 A resolution crystal structure of the C-terminal globular domain of PglB and its comparison with the previously determined structure from the archaeon Pyrococcus AglB. The two distantly related oligosaccharyltransferases share unexpected structural similarity beyond that expected from the sequence comparison. The common architecture of the putative catalytic sites revealed a new catalytic motif in PglB. Site-directed mutagenesis analyses confirmed the contribution of this motif to the catalytic function. Bacterial PglB and archaeal AglB constitute a protein family of the catalytic subunit of OST along with STT3 from eukaryotes. A structure-aided multiple sequence alignment of the STT3/PglB/AglB protein family revealed three types of OST catalytic centers. This novel classification will provide a useful framework for understanding the enzymatic properties of the OST enzymes from Eukarya, Archaea, and Bacteria.

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

Structural Basis for the Activation of Innate Immune Pattern-Recognition Receptor RIG-I by Viral RNA

The human snRNA genes transcribed by RNA polymerase II (pol II) and III (pol III) have different core promoter elements. Both gene types contain similar proximal sequence elements (PSEs) but differ in the absence (pol II) or presence (pol III) of a TATA-box, which, together with the PSE, determines the assembly of a pol III-specific pre-initiation complex. BRFU is a factor exclusively required for transcription of the pol III-type snRNA genes. We report that recruitment of BRFU to the TATA-box of these promoters is TATA-binding protein (TBP)-dependent. BRFU in turn stabilizes TBP on TATA-containing template and extends the TBP footprint both upstream and downstream of the TATA element. The core domain of TBP is sufficient for BRFU.TBP.DNA complex formation and for interaction with BRFU off the template. We have mapped amino acid residues within TBP and domains of BRFU that mediate this interaction. BRFU has no specificity for sequences flanking the TATA-box and also forms a stable complex on the TATA-box of the pol II-specific adenovirus major late promoter (AdMLP). Furthermore, pol III-type transcription can initiate from an snRNA gene promoter containing an AdMLP TATA-box and flanking sequences. Therefore, the polymerase recruitment is not simply determined by the sequence of the TATA-box and immediate flanking sequences.

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.

UmuD(2) cleaves and removes its N-terminal 24 amino acids to form UmuD'(2), which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the beta processivity clamp ("beta") of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif ((14)TFPLF(18)) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the beta-binding motif variant bind to beta with similar K(d) values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in beta is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic.

p53 protein activity as a transcription factor can be activated in vivo by antibodies that target its C-terminal negative regulatory domain suggesting that cellular enzymes that target this domain may play a role in stimulating p53-dependent gene expression. A phospho-specific monoclonal antibody to the C-terminal Ser(315) phospho-epitope was used to determine whether phosphorylation of endogenous p53 at Ser(315) can be detected in vivo, whether steady-state Ser(315) phosphorylation increases or decreases in an irradiated cell, and whether this phosphorylation event activates or inhibits p53 in vivo. A native phospho-specific IgG binding assay was developed for quantitating the extent of p53 phosphorylation at Ser(315) where one, two, three, or four phosphates/tetramer could be defined after in vitro phosphorylation by cyclin-dependent protein kinases. Using this assay, near-stoichiometric Ser(315) phosphorylation of endogenous p53 protein was detected in vivo after UV irradiation of MCF7 and A375 cells, coinciding with elevated p53-dependent transcription. Transfection of the p53 gene with an alanine mutation at the Ser(315) site into Saos-2 cells gave rise to a form of p53 protein with a substantially reduced specific activity as a transcription factor. The treatment of cells with the cyclin-dependent protein kinase inhibitor Roscovitine promoted a reduction in the specific activity of endogenous p53 or ectopically expressed p53. These results indicate that the majority of p53 protein has been phosphorylated at Ser(315) after irradiation damage and identify a cyclin-dependent kinase pathway that plays a role in stimulating p53 function.

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.

pp125(FAK) and CAKbeta/Pyk2/CadTK/RAFTK are related protein-tyrosine kinases. It is therefore of interest whether CAKbeta shares some of the properties of pp125(FAK). Using recombinant glutathione S-transferase fusion proteins, we show that the C-terminal domains of both proteins bind paxillin in vitro. The C-terminal domain of CAKbeta was engineered to be autonomously expressed in chicken embryo cells and, like pp125(FAK) and p41/43(FRNK) (the C-terminal noncatalytic domain of pp125(FAK)), was found to localize to cellular focal adhesions. In contrast, full-length CAKbeta was generally found diffusely distributed throughout the cell, although a fraction of the cells exhibited focal adhesion localization. Vanadate treatment of pp125(FAK)- and CAKbeta-overexpressing CE cells induced a dramatic increase in the phosphotyrosine content of a common set of proteins including tensin, paxillin, and p130(Cas), but some of these substrates, particularly p130(Cas), appeared to be differentially phosphorylated by pp125(FAK) and CAKbeta. Levels of tyrosine phosphorylation were higher in CAKbeta-overexpressing cells, and additional phosphotyrosine-containing species were specifically immunoprecipitated. In addition, vanadate treatment of CE cells overexpressing CAKbeta, but not pp125(FAK) overexpressors, induced a profound morphological change, which could be a consequence of the observed differences in substrate phosphorylation.

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.

Unique Matrix Structure in the Rough Endoplasmic Reticulum Cisternae of Pseudoachondroplasia Chondrocytes

Head Module Control of Mediator Interactions

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.