ABSTRACT
The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.
Subject(s)
Cloning, Molecular/methods , Prokaryotic Cells/metabolism , Proteomics/trends , Amino Acid Sequence , Automation , Base Sequence , Escherichia coli/metabolism , Europe , Fermentation , Gene Deletion , Gene Library , Genetic Vectors , Molecular Sequence Data , Protein Folding , Sequence Analysis/instrumentation , Sequence Analysis/methodsABSTRACT
The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism.
Subject(s)
Aspartate-tRNA Ligase/chemistry , RNA, Transfer, Asp/chemistry , RNA, Transfer, Asp/physiology , Base Sequence , Binding Sites , Cell Division , Crystallography, X-Ray , Escherichia coli/cytology , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Deletion , Hydrogen Bonding , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Protein Subunits , RNA, Transfer, Asp/genetics , Saccharomyces cerevisiae/genetics , Species SpecificityABSTRACT
Several crystal structures of human estrogen receptor alpha ligand-binding domain (hERalpha LBD) complexed with agonist or antagonist molecules have previously been solved. The proteins had been modified in cysteine residues (carboxymethylation) or renatured in urea to circumvent aggregation and denaturation problems. In this work, high-level protein expression and purification together with crystallization screening procedure yielded high amounts of soluble protein without renaturation or modifications steps. The native protein crystallizes in the space group P3(2) 21 with three molecules in the asymmetric unit. The overall structure is very similar to that previously reported for the hERalpha LBD with cysteine carboxymethylated residues thus validating the modification approach. The present strategy can be adapted to other cases where the solubility and the proper folding is a difficulty.
Subject(s)
Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Receptors, Estrogen/chemistry , Receptors, Estrogen/isolation & purification , Cloning, Molecular , Computer Simulation , Crystallization , Crystallography, X-Ray , Dimerization , Estrogen Receptor alpha , Humans , Ligands , Models, Molecular , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Folding , Protein Structure, Secondary/genetics , Protein Structure, Tertiary/genetics , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
The crystal structure of a triple cysteine to serine mutant ERalpha ligand-binding domain (LBD), complexed with estradiol, shows that despite the presence of a tightly bound agonist ligand, the protein exhibits an antagonist-like conformation, similar to that observed in raloxifen and 4-hydroxytamoxifen-bound structures. This mutated receptor binds estradiol with wild type affinity and displays transcriptional activity upon estradiol stimulation, but with limited potency (about 50%). This partial activity is efficiently repressed in antagonist competition assays. The comparison with available LBD structures reveals key features governing the positioning of helix H12 and highlights the importance of cysteine residues in promoting an active conformation. Furthermore the present study reveals a hydrogen bond network connecting ligand binding to protein trans conformation. These observations support a dynamic view of H12 positioning, where the control of the equilibrium between two stable locations determines the partial agonist character of a given ligand.
Subject(s)
Receptors, Estrogen/chemistry , Receptors, Estrogen/metabolism , Cloning, Molecular , Crystallography, X-Ray , Estrogen Antagonists/pharmacology , Estrogen Receptor alpha , Humans , Hydrogen Bonding , Ligands , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Estrogen/agonists , Receptors, Estrogen/geneticsABSTRACT
The crystal structures of aspartyl-tRNA synthetase (AspRS) from Thermus thermophilus, a prokaryotic class IIb enzyme, complexed with tRNA(Asp) from either T. thermophilus or Escherichia coli reveal a potential intermediate of the recognition process. The tRNA is positioned on the enzyme such that it cannot be aminoacylated but adopts an overall conformation similar to that observed in active complexes. While the anticodon loop binds to the N-terminal domain of the enzyme in a manner similar to that of the related active complexes, its aminoacyl acceptor arm remains at the entrance of the active site, stabilized in its intermediate conformational state by non-specific interactions with the insertion and catalytic domains. The thermophilic nature of the enzyme, which manifests itself in a very low kinetic efficiency at 17 degrees C, the temperature at which the crystals were grown, is in agreement with the relative stability of this non-productive conformational state. Based on these data, a pathway for tRNA binding and recognition is proposed.
Subject(s)
Aspartate-tRNA Ligase/chemistry , Aspartate-tRNA Ligase/metabolism , RNA, Bacterial/metabolism , RNA, Transfer, Asp/metabolism , Thermus thermophilus/enzymology , Thermus thermophilus/genetics , Anticodon/chemistry , Anticodon/genetics , Anticodon/metabolism , Aspartate-tRNA Ligase/genetics , Base Sequence , Binding Sites , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/genetics , Hydrogen Bonding , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Transfer, Asp/chemistry , RNA, Transfer, Asp/genetics , Structure-Activity Relationship , TemperatureABSTRACT
The 2.4 A crystal structure of the Escherichia coli aspartyl-tRNA synthetase (AspRS)-tRNA(Asp)-aspartyl-adenylate complex shows the two substrates poised for the transfer of the aspartic acid moiety from the adenylate to the 3'-hydroxyl of the terminal adenosine of the tRNA. A general molecular mechanism is proposed for the second step of the aspartylation reaction that accounts for the observed conformational changes, notably in the active site pocket. The stabilization of the transition state is mediated essentially by two amino acids: the class II invariant arginine of motif 2 and the eubacterial-specific Gln231, which in eukaryotes and archaea is replaced by a structurally non-homologous serine. Two archetypal RNA-protein modes of interactions are observed: the anticodon stem-loop, including the wobble base Q, binds to the N-terminal beta-barrel domain through direct protein-RNA interactions, while the binding of the acceptor stem involves both direct and water-mediated hydrogen bonds in an original recognition scheme.
Subject(s)
Aspartate-tRNA Ligase/chemistry , Aspartate-tRNA Ligase/metabolism , Escherichia coli/enzymology , RNA, Transfer, Amino Acyl/biosynthesis , RNA, Transfer, Asp/chemistry , RNA, Transfer, Asp/metabolism , Anticodon/chemistry , Base Sequence , Binding Sites , Crystallography, X-Ray , Escherichia coli/genetics , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Protein Structure, Secondary , RNA, Bacterial/chemistry , RNA, Bacterial/metabolismABSTRACT
The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3. When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele. The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities. Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding. This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis. Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.
Subject(s)
Aspartate-tRNA Ligase/genetics , Escherichia coli/genetics , Models, Molecular , Mutation , Structure-Activity Relationship , TemperatureABSTRACT
Escherichia coli tRNA(Asp) was overproduced in E. coli up to 15-fold from a synthetic tRNA(Asp) gene placed in a plasmid under the dependence of an isopropyl-beta,D-thiogalactopyranoside-inducible promoter. Purification to nearly homogeneity (95%) was achieved after two HPLC DEAE-cellulose columns. E. coli tRNA(Asp)[G34] (having guanine instead of queuine at position 34) was obtained by the same procedure except that it was overproduced in a strain lacking the enzyme responsible for queuine modification. Nucleoside analysis showed that, except for the replacement of Q34 by G34 in mutant-derived tRNA(Asp), the base modification levels of both tRNAs are the same as those in wild-type E. coli tRNA(Asp). Kinetic properties of tRNA(Asp)[Q34] and [G34] with yeast AspRS compared to those in the homologous reactions in yeast and E. coli clearly indicate that the major identity elements are the same in both organisms: the conserved discriminant base and the anticodon triplet. In connection with this, we explored by site-directed mutagenesis the functional role of the interactions which, as revealed by the crystallographic structure, occur between the wobble base of yeast tRNA(Asp) and two residues of yeast AspRS. Their absence strongly affected aspartylation and the kd of tRNA(Asp). Each contact individually restores almost completely the wild-type acylation properties of the enzyme; thus, wobble base recognition in yeast appears to be more protected against mutational events than in E. coli, where only one contact is thought to occur at position 34.
Subject(s)
Escherichia coli/metabolism , Guanine/analogs & derivatives , RNA, Transfer, Asp/metabolism , Transfer RNA Aminoacylation , Anticodon , Aspartate-tRNA Ligase/metabolism , Base Composition , Base Sequence , Cloning, Molecular , Crystallography, X-Ray , Guanine/chemistry , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer, Asp/chemistry , Saccharomyces cerevisiae/metabolism , Structure-Activity RelationshipABSTRACT
Crystals of the dimeric aspartyl-tRNA synthetase from Escherichia coli (molecular mass 132,000 Da) complexed with its cognate tRNA (molecular mass 25,000 Da) have been grown using ammonium sulfate as precipitant. The crystals belong to the orthorhombic space group C222(1) with unit cell parameters a = 102.75 A, b = 128.11 A, c = 231.70 A and diffract to 3 A. The asymmetric unit contains one monomer of the aspartyl-tRNA synthetase and one tRNA molecule.