Mutations in the small subunit of ribulosebisphosphate carboxylase affect subunit binding and catalysis

Fully functional Synechococcus PCC 6301 ribulose 1,5-bisphosphate carboxylase-oxygenase (kcat = 11.8 s-1) was assembled in vitro following separate expression of the large- and small-subunit genes in different Escherichia coli cultures. The small subunits were expressed predominantly as monomers, in contrast to the large subunits which have been shown to be largely octameric when expressed separately [Andrews, T. J. (1988) J. Biol. Chem. 263, 12213-12219]. This separate expression system was applied to the study of mutations in the amino-terminal arm of the small subunit, which is one of the major sites of contact with the large subunit in the assembled hexadecamer. It enabled the effects of a mutation on the tightness of binding of the small subunit to the large-subunit octamer to be distinguished from the effects of the same mutation on catalysis carried out by the assembled complex when fully saturated with mutant small subunits. This important distinction cannot be made when both subunits are expressed together in the same cell. Substitutions of conserved amino acid residues at positions 14 (Ala, Val, Gly, or Asp instead of Thr) and 17 (Cys instead of Tyr), which make important contacts with conserved large-subunit residues, were introduced by site-directed mutagenesis. All mutant small subunits were able to bind to large subunits and form active enzymes. A potential intersubunit hydrogen bond involving the Thr-14 hydroxyl group is shown to be unimportant. However, the binding of Gly-14, Asp-14, and Cys-17 mutant small subunits was weaker, and the resultant mutant enzymes had reduced catalytic rates compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changed properties of the A subunit in DNA gyrase with a B subunit mutation

Summary To investigate the interaction of subunits A and B of DNA gyrase during DNA supercoiling, a Cou^r mutant of Escherichia coli was obtained and the effect of nalidixic acid on the supercoiling of DNA by wild-type and mutant enzymes was assayed. The enzyme of the Cou^r strain proved to be more sensitive to nalidixic acid than the wild-type DNA gyrase. Hence the mutation affecting the B subunit can also change the properties of the A subunit, which fact suggests that the two subunits of DNA gyrase are in contact during DNA supercoiling.     

Structure of the theta subunit of Escherichia coli DNA polymerase III in complex with the epsilon subunit

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits, the alpha, epsilon, and theta subunits. The theta subunit is the smallest and least understood subunit. The three-dimensional structure of theta in a complex with the unlabeled N-terminal domain of the epsilon subunit, epsilon186, was determined by multidimensional nuclear magnetic resonance spectroscopy. The structure was refined using pseudocontact shifts that resulted from inserting a lanthanide ion (Dy3+, Er3+, or Ho3+) at the active site of epsilon186. The structure determination revealed a three-helix bundle fold that is similar to the solution structures of theta in a methanol-water buffer and of the bacteriophage P1 homolog, HOT, in aqueous buffer. Conserved nuclear Overhauser enhancement (NOE) patterns obtained for free and complexed theta show that most of the structure changes little upon complex formation. Discrepancies with respect to a previously published structure of free theta (Keniry et al., Protein Sci. 9:721-733, 2000) were attributed to errors in the latter structure. The present structure satisfies the pseudocontact shifts better than either the structure of theta in methanol-water buffer or the structure of HOT. satisfies these shifts. The epitope of epsilon186 on theta was mapped by NOE difference spectroscopy and was found to involve helix 1 and the C-terminal part of helix 3. The pseudocontact shifts indicated that the helices of theta are located about 15 A or farther from the lanthanide ion in the active site of epsilon186, in agreement with the extensive biochemical data for the theta-epsilon system.     

Identification of a subunit assembly domain in the alpha subunit of Escherichia coli RNA polymerase

The alpha subunit of Escherichia coli DNA-dependent RNA polymerase is encoded by the rpoA gene and plays a major role in enzyme assembly. A set of C-terminal deletion mutations of the rpoA gene was constructed. The results of mixed reconstitution experiments in vitro, using the truncated alpha polypeptides encoded by the rpoA deletion mutants, suggest that the amino-terminal two-thirds of alpha subunit is sufficient for the formation of pseudo-core complexes containing both beta and beta' subunits.     

Tissue specificity of subunit composition and EGF-dependent changes in the subunit pattern of 20S-proteasomes

The subunit pattern of 20S proteasomes from rat kidney, rat liver, human A-431 cells, human K-562 cells and mouse NIH 3T3 cells were studied. Proteasomes in cells of a common tissue origin appeared to be similar, independently of the intensity of cell proliferation. Unlike, proteasomes in cells of various types of tissue specificity differed from each other. Besides, EGF was shown to induce changes in the subunit pattern of proteasomes in A-431 cells.     

Purification of the large ribosomal subunit via its association with the small subunit

We have developed an affinity purification of the large ribosomal subunit from Deinococcus radiodurans that exploits its association with FLAG-tagged 30S subunits. Thus, capture is indirect so that no modification of the 50S is required and elution is achieved under mild conditions (low magnesium) that disrupt the association, avoiding the addition of competitor ligands or coelution of common contaminants. Efficient purification of highly pure 50S is achieved, and the chromatography simultaneously sorts the 50S into three classes according to their association status (unassociated, loosely associated, or tightly associated), improving homogeneity.     

Regulation of the acid-labile subunit in sustained endotoxemia

The effect of sustained endotoxemia on expression of the acid-labile subunit (ALS) in relation to hepatic markers of altered GH and insulin sensitivity was examined. Juvenile rats were injected with endotoxin twice daily for 48 h, causing reduced food intake and attenuated growth. In pair-fed controls, food restriction caused marked suppression of ALS gene expression and circulating levels within 12 h, and endotoxemia augmented this effect. This acute effect of endotoxin corresponded temporally with transient induction of suppressor of cytokine signaling (SOCS)-3, cytokine-inducible SH2-containing protein (CIS), phosphoenolpyruvate carboxykinase (PEPCK), and insulin-like growth factor-binding protein (IGFBP)-1 and suppression of GH receptor (GHR). During the subsequent 36 h of sustained endotoxin treatment, expression of ALS recovered to, and then rose above, that of their pair-fed controls. This effect was paralleled by other ternary complex components. The inductive effect of sustained endotoxemia relative to pair-fed controls could not be explained by differences in expression of GHR, SOCS-3, or CIS but coincided with normalized PEPCK and IGFBP-1 levels, suggesting better hepatic insulin sensitivity in these animals. These data may indicate that, in sustained endotoxemia, ALS levels are regulated through modulation of hepatic insulin sensitivity.     

Site-specific cleavage of the 40S ribosomal subunit reveals eukaryote-specific ribosomal protein S28 in the subunit head

After resolving the crystal structure of the prokaryotic ribosome, mapping the proteins in the eukaryotic ribosome is a challenging task. We applied RNase H digestion to split the human 40S ribosomal subunit into head and body parts. Mass spectrometry of the proteins in the 40S subunit head revealed the presence of eukaryote-specific ribosomal protein S28e. Recombinant S28e was capable of specific binding to the 3′ major domain of the 18S rRNA (K_a = 8.0 ± 0.5 × 10⁹ M⁻¹). We conclude that S28e has a binding site on the 18S rRNA within the 40S subunit head.

Structured summary

MINT-8044084: S8 (uniprotkb:P62241) and S19 (uniprotkb:P39019) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044095: S8 (uniprotkb:P62241), S19 (uniprotkb:P39019) and S13 (uniprotkb:P62277) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044024: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S21 (uniprotkb:P63220), S20 (uniprotkb:P60866), S26 (uniprotkb:P62854), S25 (uniprotkb:P62851), S12 (uniprotkb:P25398), S17 (uniprotkb:P08708), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263), S16 (uniprotkb:P62249) and S11 (uniprotkb:P62280) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-8044065: S29 (uniprotkb:P62273), S28 (uniprotkb:P62857), S19 (uniprotkb:P39019), S14 (uniprotkb:P62263) and S16 (uniprotkb:P62249) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V(1) sector and entire V(1)V(O) complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G(1-59)) of the V(1)V(O) ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of G(1-59) in the absence and presence of the N-terminal peptides E(1-18) and E(18-38) as well as the produced and purified C-terminal segment (E(39-233)) shows specific interactions only with the peptide fragment E(18-38). The binding of this peptide occurs via the residues M(1), V(2), S(3), and K(5) as well for V(22), S(23), K(24), A(25) and R(26) of G(1-59). The specific E(18-38)/G(1-59) binding has been confirmed by fluorescence correlation spectroscopy data. The E(18-38) peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E(18-38)/G(1-59) complex reveals the orientation of E(18-38) relative to G(1-59) via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.     

Serotonin induces selective cleavage of the PKA RI subunit but not RII subunit in Aplysia neurons

PKA type I and type II are activated in Aplysia neurons by stimulation with serotonin (5-HT), which causes long-term facilitation (LTF). The proteolysis of the regulatory subunit (R) is thought important for the persistent activation of PKA, which is necessary to produce LTF. In this study, we report that the type I regulatory subunit (RI) and type II regulatory subunit (RII) are differentially regulated by proteolytic cleavage. RI, but not RII, was selectively cleaved after 5-HT treatment for 2h in Aplysia neurons. Interestingly, the proteasome inhibitor MG132 inhibited the cleavage of RI caused by 5-HT treatment in Aplysia neuron. Besides extracts from Aplysia ganglia treated with 5-HT cleaved (35)S-labeled RI synthesized in vitro, but not (35)S-labeled RII. This suggests that 5-HT induces the activation state of RI-specific proteolytic cleavage.     

Advancements in the development of subunit influenza vaccines

The ongoing threat of influenza epidemics and pandemics has emphasized the importance of developing safe and effective vaccines against infections from divergent influenza viruses. In this review, we first introduce the structure and life cycle of influenza A viruses, describing major influenza A virus-caused pandemics. We then compare different types of influenza vaccines and discuss current advancements in the development of subunit influenza vaccines, particularly those based on nucleoprotein (NP), extracellular domain of matrix protein 2 (M2e) and hemagglutinin (HA) proteins. We also illustrate potential strategies for improving the efficacy of subunit influenza vaccines.     

Analysis of the subunit stoichiometries in viral entry

Virions of the Human Immunodeficiency Virus (HIV) infect cells by first attaching with their surface spikes to the CD4 receptor on target cells. This leads to conformational changes in the viral spikes, enabling the virus to engage a coreceptor, commonly CCR5 or CXCR4, and consecutively to insert the fusion peptide into the cellular membrane. Finally, the viral and the cellular membranes fuse. The HIV spike is a trimer consisting of three identical heterodimers composed of the gp120 and gp41 envelope proteins. Each of the gp120 proteins in the trimer is capable of attaching to the CD4 receptor and the coreceptor, and each of the three gp41 units harbors a fusion domain. It is still under debate how many of the envelope subunits within a given trimer have to bind to the CD4 receptors and to the coreceptors, and how many gp41 protein fusion domains are required for fusion. These numbers are referred to as subunit stoichiometries. We present a mathematical framework for estimating these parameters individually by analyzing infectivity assays with pseudotyped viruses. We find that the number of spikes that are engaged in mediating cell entry and the distribution of the spike number play important roles for the estimation of the subunit stoichiometries. Our model framework also shows why it is important to subdivide the question of the number of functional subunits within one trimer into the three different subunit stoichiometries. In a second step, we extend our models to study whether the subunits within one trimer cooperate during receptor binding and fusion. As an example for how our models can be applied, we reanalyze a data set on subunit stoichiometries. We find that two envelope proteins have to engage with CD4-receptors and coreceptors and that two fusion proteins must be revealed within one trimer for viral entry. Our study is motivated by the mechanism of HIV entry but the experimental technique and the model framework can be extended to other viral systems as well.     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V₁ sector and entire V₁V_O complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G_1–59) of the V₁V_O ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of G_1–59 in the absence and presence of the N-terminal peptides E_1–18 and E_18–38 as well as the produced and purified C-terminal segment (E_39–233) shows specific interactions only with the peptide fragment E_18–38. The binding of this peptide occurs via the residues M₁, V₂, S₃, and K₅ as well for V₂₂, S₂₃, K₂₄, A₂₅ and R₂₆ of G_1–59. The specific E_18–38/G_1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E_18–38 peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E_18–38/G_1–59 complex reveals the orientation of E_18–38 relative to G_1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.     

The light subunit of system b(o,+) is fully functional in the absence of the heavy subunit

The heteromeric amino acid transporters are composed of a type II glycoprotein and a non-glycosylated polytopic membrane protein. System b(o,+) exchanges dibasic for neutral amino acids. It is composed of rBAT and b(o,+)AT, the latter being the polytopic membrane subunit. Mutations in either of them cause malfunction of the system, leading to cystinuria. b(o,+)AT-reconstituted systems from HeLa or MDCK cells catalysed transport of arginine that was totally dependent on the presence of one of the b(o,+) substrates inside the liposomes. rBAT was essential for the cell surface expression of b(o,+)AT, but it was not required for reconstituted b(o,+)AT transport activity. No system b(o,+) transport was detected in liposomes derived from cells expressing rBAT alone. The reconstituted b(o,+)AT showed kinetic asymmetry. Expressing the cystinuria-specific mutant A354T of b(o,+)AT in HeLa cells together with rBAT resulted in defective arginine uptake in whole cells, which was paralleled by the reconstituted b(o,+)AT activity. Thus, subunit b(o,+)AT by itself is sufficient to catalyse transmembrane amino acid exchange. The polytopic subunits may also be the catalytic part in other heteromeric transporters.     

Nitric oxide activates the beta 2 subunit of soluble guanylyl cyclase in the absence of a second subunit.

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.     

An algal nucleus-encoded subunit of mitochondrial ATP synthase rescues a defect in the analogous human mitochondrial-encoded subunit

Unlike most organisms, the mitochondrial DNA (mtDNA) of Chlamydomonas reinhardtii, a green alga, does not encode subunit 6 of F(0)F(1)-ATP synthase. We hypothesized that C. reinhardtii ATPase 6 is nucleus encoded and identified cDNAs and a single-copy nuclear gene specifying this subunit (CrATP6, with eight exons, four of which encode a mitochondrial targeting signal). Although the algal and human ATP6 genes are in different subcellular compartments and the encoded polypeptides are highly diverged, their secondary structures are remarkably similar. When CrATP6 was expressed in human cells, a significant amount of the precursor polypeptide was targeted to mitochondria, the mitochondrial targeting signal was cleaved within the organelle, and the mature polypeptide was assembled into human ATP synthase. In spite of the evolutionary distance between algae and mammals, C. reinhardtii ATPase 6 functioned in human cells, because deficiencies in both cell viability and ATP synthesis in transmitochondrial cell lines harboring a pathogenic mutation in the human mtDNA-encoded ATP6 gene were overcome by expression of CrATP6. The ability to express a nucleus-encoded version of a mammalian mtDNA-encoded protein may provide a way to import other highly hydrophobic proteins into mitochondria and could serve as the basis for a gene therapy approach to treat human mitochondrial diseases.     

Binding of erythromycin to the 50S ribosomal subunit is affected by alterations in the 30S ribosomal subunit

Summary Expression of resistance to erythromycin in Escherichia coli, caused by an altered L4 protein in the 50S ribosomal subunit, can be masked when two additional ribosomal mutations affecting the 30S proteins S5 and S12 are introduced into the strain (Saltzman, Brown, and Apirion, 1974). Ribosomes from such strains bind erythromycin to the same extent as ribosomes from erythromycin sensitive parental strains (Apirion and Saltzman, 1974).Among mutants isolated for the reappearance of erythromycin resistance, kasugamycin resistant mutants were found. One such mutant was analysed and found to be due to undermethylation of the rRNA. The ribosomes of this strain do not bind erythromycin, thus there is a complete correlation between phenotype of cells with respect to erythromycin resistance and binding of erythromycin to ribosomes.Furthermore, by separating the ribosomal subunits we showed that 50S ribosomes bind or do not bind erythromycin according to their L4 protein; 50S with normal L4 bind and 50S with altered L4 do not bind erythromycin. However, the 30s ribosomes with altered S5 and S12 can restore binding in resistant 50S ribosomes while the 30S ribosomes in which the rRNA also became undermethylated did not allow erythromycin binding to occur.Thus, evidence for an intimate functional relationship between 30S and 50S ribosomal elements in the function of the ribosome could be demonstrated. These functional interrelationships concerns four ribosomal components, two proteins from the 30S ribosomal subunit, S5, and S12, one protein from the 50S subunit L4, and 16S rRNA.