177,162 research outputs found
Overproduction, purification, crystallization and preliminary X-ray diffraction studies of the human spliceosomal protein U5-15kD
No full textThe gene coding for the human spliceosomal U5 snRNP-specific 15 kDa protein (U5-15kD) was overexpressed in Escherichia coli, its product purified to homogeneity and crystallized. Well diffracting single crystals were obtained by the vapour-diffusion method in hanging drops and subsequent macroseeding, The crystals belong to the orthorhombic space group P2(1)2(1)2 with a = 62.3, b = 65.7, c = 37.1 Angstrom. They diffract to at least 3.0 Angstrom and contain one molecule in the asymmetric unit. A selenomethionine derivative of the protein was prepared and crystallized for multiwavelength anomalous diffraction (MAD) data collection
Crystal structure of a subtilisin-like serine proteinase from a psychrotrophic Vibrio species reveals structural aspects of cold adaptation
No full textThe crystal structure of a subtilisin-like serine proteinase from the psychrotrophic marine bacterium, Vibrio sp. PA-44, was solved by means of molecular replacement and refined at 1.84 Angstrom. This is the first structure of a cold-adapted subtilase to be determined and its elucidation facilitates examination of the molecular principles underlying temperature adaptation in enzymes. The cold-adapted Vibrio proteinase was compared with known three-dimensional structures of homologous enzymes of meso- and thermophilic origin, proteinase K and thermitase, to which it has high structural resemblance. The main structural features emerging as plausible determinants of temperature adaptation in the enzymes compared involve the character of their exposed and buried surfaces, which may be related to temperature-dependent variation in the physical properties of water. Thus, the hydrophobic effect is found to play a significant role in the structural stability of the meso- and thermophile enzymes, whereas the cold-adapted enzyme has more of its apolar surface exposed. In addition, the cold-adapted Vibrio proteinase is distinguished from the more stable enzymes by its strong anionic character arising from the high occurrence of uncompensated negatively charged residues at its surface. Interestingly, both the cold-adapted and thermophile proteinases differ from the mesophile enzyme in having more extensive hydrogen- and ion pair interactions in their structures; this supports suggestions of a dual role of electrostatic interactions in the adaptation of enzymes to both high and low temperatures. The Vibrio proteinase has three calcium ions associated with its structure, one of which is in a calcium-binding site not described in other subtilases
eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining
No full texteIF5B is a eukaryal translational GTPase that catalyzes ribosomal subunit joining to form elongation‐competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of eIF5B or its archaeal and bacterial (IF2) orthologs remained unclear. Here, we present six high‐resolution crystal structures of eIF5B in its apo, GDP‐ and GTP‐bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by eIF5B. Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTPase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP‐bound eIF5B. Based on these data, we propose a novel domain release mechanism for eIF5B/IF2 activation that explains how eIF5B and IF2 fulfill their catalytic role during ribosomal subunit joining
A monovalent cation acts as structural and catalytic cofactor in translational GTPases
No full textTranslational GTPases are universally conserved GTP hydrolyzing enzymes, critical for fidelity and speed of ribosomal protein biosynthesis. Despite their central roles, the mechanisms of GTP‐dependent conformational switching and GTP hydrolysis that govern the function of trGTPases remain poorly understood. Here, we provide biochemical and high‐resolution structural evidence that eIF5B and aEF1A/EF‐Tu bound to GTP or GTPγS coordinate a monovalent cation (M) in their active site. Our data reveal that M ions form constitutive components of the catalytic machinery in trGTPases acting as structural cofactor to stabilize the GTP‐bound “on” state. Additionally, the M ion provides a positive charge into the active site analogous to the arginine‐finger in the Ras‐RasGAP system indicating a similar role as catalytic element that stabilizes the transition state of the hydrolysis reaction. In sequence and structure, the coordination shell for the M ion is, with exception of eIF2γ, highly conserved among trGTPases from bacteria to human. We therefore propose a universal mechanism of M‐dependent conformational switching and GTP hydrolysis among trGTPases with important consequences for the interpretation of available biochemical and structural data
Sequence analysis and overexpression of the Zymomonas mobilis tgt gene encoding tRNA-guanine transglycosylase: purification and biochemical characterization of the enzyme
No full texttRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration.tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q). It catalyzes the posttranscriptional base exchange of the Q precursor 7-aminomethyl-7-deazaguanine (preQ1) with the genetically encoded guanine in the anticodon of tRNA(Asp), tRNA(Asn), tRNA(His), and tRNA(Tyr). A partially sequenced gene upstream of the DNA ligase (lig) gene of the Zymomonas mobilis chromosome shows strong homology to the tgt gene of Escherichia coli (K.B. Shark and T. Conway, FEMS Microbiol. Lett. 96:19-26, 1992). We showed that this gene is able to complement the tgt mutation in E. coli SJ1505, and we determined its complete sequence. Four start codons were possible for this gene, resulting in proteins of 386 to 399 amino acids (M(r), 42,800 to 44,300) showing 60.4% sequence identity with Tgt from E. coli. The smallest of the four possible reading frames, which was still extended at its 5' end compared with the E. coli tgt gene, was overexpressed in E. coli. The gene product was purified to homogeneity and was biochemically characterized. The kinetical parameters were virtually identical to those published for the E. coli enzyme. In contrast to E. coli Tgt, which is reported to be a homotrimer, Z. mobilis Tgt was found to be a monomer according to gel filtration. In this study, it was shown that the formation of homotrimers by the E. coli enzyme is readily reversible and is dependent on protein concentration
Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography
No full textLakomek K, Dickmanns A, Kettwig M, Urlaub H, Ficner R, Lübke T. Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography. BMC Structural Biology. 2009;9(1):56-72
Structural basis for m(3)G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1
No full textIn higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m(7) G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m(7)G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m(3)G)-cap. The import adaptor snurportin1 recognises the m(3)G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m(3)G-cap-binding domain of snurportin1 with bound m(3)GpppG at 2.4 angstrom resolution, revealing a structural similarity to the mRNA-guanylytransferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m(7)G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m(3)G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m(7)G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants
Crystallization and preliminary crystallographic studies of Sfp: a phosphopantetheinyl transferase of modular peptide synthetases
No full textThe Bacillus subtilis Sfp protein is required for the non-ribosomal biosynthesis of the lipoheptapeptide antibiotic surfactin. It converts seven peptidyl carrier protein (PCP) domains of the surfactin synthetase SfrA-(A-C) to their active hole-forms by 4'-phosphopantetheinylation. The B. subtilis sfp gene was overexpressed in Escherichia coli and its gene product was purified to homogeneity and crystallized. Well diffracting single crystals were obtained from Sfp as well as from a selenomethionyl derivative, using sodium formate as a precipitant. The crystals belong to the tetragonal space group P4(1)2(1)2/P4(3)2(1)2, with unit-cell parameters a = b = 65.3, c = 150.5 Angstrom. They diffract beyond 2.8 Angstrom and contain one molecule in the asymmetric unit
3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: biological significance, three-dimensional structure and gene regulation
No full text3 alpha -Hydroxysteroid dehydrogenase/carbonyl reductase (3 alpha -HSD/CR) catalyses the oxidoreduction at carbon 3 of steroid hormones and is postulated to initiate the complete mineralisation of the steroid nucleus to CO2 and H2O in Comamanas testosteroni. The enzyme was found to be functional towards a variety of steroid substrate, including the steroid antibiotic fusidic acid. The enzyme also catalyses the carbonyl reduction of nonsteroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3 alpha -HSD/CR contributes to important defense strategies of C. testosteroni against natural and synthetic toxicants. The 3 alpha -HSD/CR gene (hsdA) is 774 base pairs long and the deduced amino acid sequence comprises 258 residues with a calculated molecular mass of 26.4 kDa. A homology search revealed 3 alpha -HSD/CR as a new member of the short-chain dehydro genase/reductase (SDR) superfamily, Upon gel permeation chromatography the purified enzyme elutes as a 49.4 kDa protein indicating a dimeric nature of 3 alpha -HSD/CR. The protein was crystallised and the structure solved by X-ray analysis. The crystal structure reveals one homodimer per asymmetric unit, thereby verifying its dimeric nature. Dimerisation takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far, this type of intermolecular contact has exclusively been observed in homotetrameric SDRs, but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3 alpha -HSD/CR by the presence of a predominantly alpha -helical subdomain, which is missing in all other SDRs of known structure. The promoter domain was localised within the 93 bp region upstream of hsdA and the transcriptional start site was identified at 28 bp upstream of the translation start site. Interestingly, hsdA expression was found to be under negative control by two repressor proteins, the genes of which were found in opposite direction downstream or overlapping with hsdA. Based on our results, we propose that induction of hsdA expression in C. testosteroni by steroids actually appears to be a de-repression by preventing the binding of repressor proteins to regulatory regions. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved
Structure of the novel alpha amylase AmyC from Thermotoga maritima
No full textalpha-Amylases are essential enzymes in alpha-glucan metabolism and catalyse the hydrolysis of long sugar polymers such as amylose and starch. The crystal structure of a previously unidentified amylase (AmyC) from the hyperthermophilic organism Thermotoga maritima was determined at 2.2 angstrom resolution by means of MAD. AmyC lacks sequence similarity to canonical alpha-amylases, which belong to glycosyl hydrolase families 13, 70 and 77, but exhibits significant similarity to a group of as yet uncharacterized proteins in COG1543 and is related to glycerol hydrolase family 57 (GH-57). AmyC reveals features that are characteristic of alpha-amylases, such as a distorted TIM-barrel structure formed by seven beta-strands and alpha-helices ( domain A), and two additional but less well conserved domains. The latter are domain B, which contains three helices inserted in the TIM-barrel after beta-sheet 2, and domain C, a five-helix region at the C-terminus. Interestingly, despite moderate sequence homology, structure comparison revealed significant similarities to a member of GH-57 with known three-dimensional structure, Thermococcus litoralis 4-glucanotransferase, and an even higher similarity to a structure of an enzyme of unknown function from Thermus thermophilus
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