Solution structure of Escherichia coli HypC

Escherichia coli HypC plays an important role in the maturation process of the pre-maturated HycE, the large subunit of hydrogenase 3. It serves as an iron transfer as well as a chaperone protein during the maturation process of pre-HycE, and interacts with both HypD and HycE. The N-terminal cysteine residue of HypC plays a key role in the protein-protein interactions. Here, we present the three-dimensional structure of E. coli HypC, the first solution structure of HupF/HypC family. Our result demonstrates that E. coli HypC consists of a typical OB-fold beta-barrel with two C-terminal helixes. Sequence alignment and structural comparison reveal that the hydrophobic region on the surface of E. coli HypC, as well as the highly flexible C-terminal helixes, may involve in the interactions of E. coli HypC with other proteins.     

Quantifying Escherichia coli glutaredoxin-3 substrate specificity using ligand-induced stability

Traditionally, quantification of protein-ligand affinity is performed using kinetic or equilibrium measurements. However, if the binding reaction proceeds via a stable covalent complex, these approaches are often limited. By exploiting the fact that the conformational stabilization of a protein is altered upon ligand binding due to specific interactions, and using an array of selectively chosen ligand analogs, one can quantify the contribution individual interactions have on specificity. We have used ligand-induced stability as a basis to dissect the interaction between glutaredoxin-3 (Grx3) and one of its native substrates, the tripeptide glutathione. Taking advantage of the fact that Grx3 can be trapped in a covalent mixed disulfide to glutathione or to selected synthetic glutathione analogs as part of the natural catalytic cycle, individual contributions to binding of specific molecular groups can be quantified by changes in ligand-induced stability. These changes in conformational stability are interpreted in terms of interaction energies (i.e. specificity) of the particular groups present on the ligand analog. Our results illustrate that although Grx3 recognizes glutathione predominantly through independent and additive ionic interactions at the N- and C-terminal of glutathione, van der Waals interactions from the unique gamma-glutamate moiety of glutathione also play an important role. This study places us closer to understanding the complex task of accommodating multiple substrate specificities in proteins of the thioredoxin superfamily and underscores the general applicability of ligand-induced stability to probe substrate specificity.     

Mitochondrial inner membrane protease 1 of Saccharomyces cerevisiae shows sequence similarity to the Escherichia coli leader peptidase

Summary The nuclear yeast mutant pet ts2858 is defective in the removal of pre-sequences from the mitochondrially encoded cytochrome oxidase subunit II (COXII) and the processing intermediate of cytochrome b ₂ (Cytb ₂), a nuclear gene product. In order to identify the genetic lesion in this mutant we have cloned and characterized a DNA region which complements the pet ts2858 mutation. The DNA sequence revealed three open reading frames, one of which is responsible for the complementation. A 570 by reading frame represents the structural gene PET2858, as demonstrated by in vitro mutagenesis, gene expression from a foreign promoter, and allelism tests. PET2858 encodes a 21.4 kDa protein, which is essential for growth on non-fermentable carbon sources and for the proteolytic processing of COXII and the Cytb ₂ intermediate. When the N-terminus of the PET2858 protein is fused to a reporter protein, the resulting hybrid molecule is imported into mitochondria. Interestingly, the N-terminal half of the deduced PET2858 protein exhibits 30.7% amino acid identity to the leader peptidase of Escherichia coli. These results suggest that PET2858 codes for a mitochondrial inner membrane protease (IMP1) or at least a subunit of it. This protease is involved in protein processing and export from the mitochondrial matrix.Dedicated to Professor Dr. Peter Starlinger on the occasion of his 60th birthday     

The crystal structure of the Bacillus anthracis spore surface protein BclA shows remarkable similarity to mammalian proteins

The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.     

Escherichia coli shows two types of behavioral responses to osmotic upshift.

Behavioral responses to osmotic upshift were characterized by temporal assays of free-swimming cells of Escherichia coli. Small osmotic upshifts (200 to 300 mosM) elicited tumble responses which were chemotaxis dependent, while large osmotic upshifts (400 to 500 mosM) elicited stopping followed by pseudotumbling which was chemotaxis independent.     

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.     

Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.     

The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases.

The structure of the Escherichia coli ribosomal protein L25 has been determined to an r.m.s. displacement of backbone heavy atoms of 0.62 +/- 0.14 A by multi-dimensional heteronuclear NMR spectroscopy on protein samples uniformly labeled with 15N or 15N/13C. L25 shows a new topology for RNA-binding proteins consisting of a six-stranded beta-barrel and two alpha-helices. A putative RNA-binding surface for L25 has been obtained by comparison of backbone 15N chemical shifts for L25 with and without a bound cognate RNA containing the eubacterial E-loop that is the site for binding of L25 to 5S ribosomal RNA. Sequence comparisons with related proteins, including the general stress protein, CTC, show that the residues involved in RNA binding are highly conserved, thereby providing further confirmation of the binding surface. Tertiary structure comparisons indicate that the six-stranded beta-barrels of L25 and of the tRNA anticodon-binding domain of glutaminyl-tRNA synthetase are similar.     

Solution structure of C-terminal Escherichia coli translation initiation factor IF2 by small-angle X-ray scattering

Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule. Furthermore, the SAXS data revealed that E. coli IF2 in solution adopts a structure that is significantly different from the crystal structure of orthologous aIF5B from Methanobacterium thermoautotrophicum. This crystal structure constitutes the only atomic resolution structural knowledge of the full-length factor. Computer programs were applied to the SAXS data to provide an initial structural model for IF2 in solution. The low-resolution nature of SAXS prevents the elucidation of a complete and detailed structure, but the resulting model for C-terminal E. coli IF2 indicates important structural differences between the aIF5B crystal structure and IF2 in solution. The chalice-like structure with a highly exposed alpha-helical stretch observed for the aIF5B crystal structure was not found in the structural model of IF2 in solution, in which domain VI-2 is moved closer to the rest of the protein.     

Solution structure of the sulfite reductase flavodoxin-like domain from Escherichia coli

The flavoprotein moiety of Escherichia coli sulfite reductase (SiR-FP) is homologous to electron transfer proteins such as cytochrome-P450 reductase (CPR) or nitric oxide synthase (NOS). We report on the three-dimensional structure of SiR-FP18, the flavodoxin-like domain of SiR-FP, which has been determined by NMR. In the holoenzyme, this domain plays an important role by shuttling electrons from the FAD to the hemoprotein (the beta-subunit). The structure presented here was determined using distance and torsion angle information in combination with residual dipolar couplings determined in two different alignment media. Several protein-FMN NOEs allowed us to place the prosthetic group in its binding pocket. The structure is well-resolved, and (15)N relaxation data indicate that SiR-FP18 is a compact domain. The binding interface with cytochrome c, a nonphysiological electron acceptor, has been determined using chemical shift mapping. Comparison of the SiR-FP18 structure with the corresponding domains from CPR and NOS shows that the fold of the protein core is highly conserved, but the analysis of the electrostatic surfaces reveals significant differences between the three domains. These observations are placed in the physiological context so they can contribute to the understanding of the electron transfer mechanism in the SiR holoenzyme.     

Structure of Escherichia coli AMP nucleosidase reveals similarity to nucleoside phosphorylases

AMP nucleosidase (AMN) catalyzes the hydrolysis of AMP to form adenine and ribose 5-phosphate. The enzyme is found only in prokaryotes, where it plays a role in purine nucleoside salvage and intracellular AMP level regulation. Enzyme activity is stimulated by ATP and suppressed by phosphate. The structure of unliganded AMN was determined at 2.7 A resolution, and structures of the complexes with either formycin 5'-monophosphate or inorganic phosphate were determined at 2.6 A and 3.0 A resolution, respectively. AMN is a biological homohexamer, and each monomer is composed of two domains: a catalytic domain and a putative regulatory domain. The overall topology of the catalytic domain and some features of the substrate binding site resemble those of the nucleoside phosphorylases, demonstrating that AMN is a new member of the family. The structure of the regulatory domain consists of a long helix and a four-stranded sheet and has a novel topology.     

Solution structure of psi32-modified anticodon stem-loop of Escherichia coli tRNAPhe

Nucleoside base modifications can alter the structures and dynamics of RNA molecules and are important in tRNAs for maintaining translational fidelity and efficiency. The unmodified anticodon stem–loop from Escherichia coli tRNA^Phe forms a trinucleotide loop in solution, but Mg²⁺ and dimethylallyl modification of A₃₇ N6 destabilize the loop-proximal base pairs and increase the mobility of the loop nucleotides. The anticodon arm has three additional modifications, ψ₃₂, ψ₃₉, and A₃₇ C2-thiomethyl. We have used NMR spectroscopy to investigate the structural and dynamical effects of ψ₃₂ on the anticodon stem-loop from E.coli tRNA^Phe. The ψ₃₂ modification does not significantly alter the structure of the anticodon stem–loop relative to the unmodified parent molecule. The stem of the RNA molecule includes base pairs ψ₃₂-A₃₈ and U₃₃–A₃₇ and the base of ψ₃₂ stacks between U₃₃ and A₃₁. The glycosidic bond of ψ₃₂ is in the anti configuration and is paired with A₃₈ in a Watson–Crick geometry, unlike residue 32 in most crystal structures of tRNA. The ψ₃₂ modification increases the melting temperature of the stem by ~3.5°C, although the ψ₃₂ and U₃₃ imino resonances are exchange broadened. The results suggest that ψ₃₂ functions to preserve the stem integrity in the presence of additional loop modifications or after reorganization of the loop into a translationally functional conformation.     

Solution structure of the carbon storage regulator protein CsrA from Escherichia coli

The carbon storage regulator A (CsrA) is a protein responsible for the repression of a variety of stationary-phase genes in bacteria. In this work, we describe the nuclear magnetic resonance (NMR)-based structure of the CsrA dimer and its RNA-binding properties. CsrA is a dimer of two identical subunits, each composed of five strands, a small alpha-helix and a flexible C terminus. NMR titration experiments suggest that the beta1-beta2 and beta3-beta4 loops and the C-terminal helix are important elements in RNA binding. Even though the beta3-beta4 loop contains a highly conserved RNA-binding motif, GxxG, typical of KH domains, our structure excludes CsrA from being a member of this protein family, as previously suggested. A mechanism for the recognition of mRNAs downregulated by CsrA is proposed.     

Solution structure of the bacterial chemotaxis adaptor protein CheW from Escherichia coli

The bacterial chemotaxis adaptor protein CheW physically links the chemoreceptors (MCPs) and the histidine kinase CheA. Extensive investigations using bacterium Escherichia coli have established the central role of CheW in the MCP-modulated activation of CheA. Here we report the solution structure of CheW from E. coli determined by NMR spectroscopy. The results show that E. coli CheW shares an overall fold with previously reported structure of CheW from Thermotoga maritima, whereas local conformational deviations are observed. In particular, the C-terminal alpha-helix is considerably longer in E. coli CheW and appears to shrink the active binding pocket with CheA. Our study provides the structural basis for further investigations in E. coli chemotaxis.     

Solution structure of the Escherichia coli protein ydhR: a putative mono-oxygenase

YdhR is a 101-residue conserved protein from Escherichia coli. Sequence searches reveal that the protein has >50% identity to proteins found in a variety of other bacterial genomes. Using size exclusion chromatography and fluorescence spectroscopy, we determined that ydhR exists in a dimeric state with a dissociation constant of approximately 40 nM. The three-dimensional structure of dimeric ydhR was determined using NMR spectroscopy. A total of 3400 unambiguous NOEs, both manually and automatically assigned, were used for the structure calculation that was refined using an explicit hydration shell. A family of 20 structures was obtained with a backbone RMSD of 0.48 A for elements of secondary structure. The structure reveals a dimeric alpha,beta fold characteristic of the alpha+beta barrel superfamily of proteins. Bioinformatic approaches were used to show that ydhR likely belongs to a recently identified group of mono-oxygenase proteins that includes ActVA-Orf6 and YgiN and are involved in the oxygenation of polyaromatic ring compounds.     

Structural similarity of YbeD protein from Escherichia coli to allosteric regulatory domains

Lipoic acid is an essential prosthetic group in several metabolic pathways. The biosynthetic pathway of protein lipoylation in Escherichia coli involves gene products of the lip operon. YbeD is a conserved bacterial protein located in the dacA-lipB intergenic region. Here, we report the nuclear magnetic resonance structure of YbeD from E. coli. The structure includes a beta alpha beta beta alpha beta fold with two alpha-helices on one side of a four-strand antiparallel beta-sheet. The beta 2-beta 3 loop shows the highest sequence conservation and is likely functionally important. The beta-sheet surface contains a patch of conserved hydrophobic residues, suggesting a role in protein-protein interactions. YbeD shows striking structural homology to the regulatory domain from d-3-phosphoglycerate dehydrogenase, hinting at a role in the allosteric regulation of lipoic acid biosynthesis or the glycine cleavage system.     

Solution structure of microcin J25, the single macrocyclic antimicrobial peptide from Escherichia coli.

The three-dimensional solution structure of microcin J25, the single cyclic representative of the microcin antimicrobial peptide class produced by enteric bacteria, was determined using two-dimensional 1H NMR spectroscopy and molecular modeling. This hydrophobic 21-residue peptide exhibits potent activity directed to Gram-negative bacteria. Its primary structure, cyclo(-V1GIGTPISFY10GGGAGHVPEY20F-), has been determined previously [Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R.A., Farías, R.N., Moreno, F. & Rebuffat, S. (1999) Eur. J. Biochem., 259, 747-755]. Conformational parameters (3JNHCalphaH coupling constants, quantitative nuclear Overhauser enhancement data, chemical shift deviations, temperature coefficients of amide protons, NH-ND exchange rates) were obtained in methanol solution. Structural restraints consisting of 190 interproton distances inferred from NOE data, 11 phi backbone dihedral angle and 9 chi1 angle restraints derived from the coupling constants and three hydrogen bonds in agreement with the amide exchange rates were used as input for simulated annealing calculations and energy minimization in the program XPLOR. Microcin J25 adopts a well-defined compact structure consisting of a distorted antiparallel beta sheet, which is twisted and folded back on itself, thus resulting in three loops. Residues 7-10 and 17-20 form the more regular part of the beta sheet. The region encompassing residues Gly11-His16 consists of a distorted beta hairpin, which divides into two small loops and is stabilized by an inverse gamma turn and a type I' beta turn. The reversal of the chain leading to the Phe21-Pro6 loop results from a mixed beta/gamma turn. A cavity, in which the hydrophilic Ser8 side-chain is confined, is delimited by two crab pincer-like regions that comprise residues 6-8 and 18-1.     

Penicillin-binding protein 4 of Escherichia coli shows a novel type of primary structure among penicillin-interacting proteins

The nucleotide sequence of a 1884 bp DNA fragment of E. coli, carrying the gene dacB, was determined. The DNA codes for penicillin-binding protein 4 (PBP4), an enzyme of 477 amino acids, being involved as a DD-carboxypeptidase-endopeptidase in murein metabolism. The enzyme is translated with a cleavable signal peptide of 20 amino acids, which was verified by sequencing the amino-terminus of the isolated protein. The characteristic active-site fingerprints SXXK, SXN and KTG of class A beta-lactamases and penicillin-binding proteins were located in the sequence. On the basis of amino acid alignments we propose, that PBP4 and class A beta-lactamases share a common evolutionary origin but PBP4 has acquired an additional domain of 188 amino acids in the region between the SXXK and SXN elements.