Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride

The aim of this work was to study the conformational changes of the Escherichia coli glutamine-binding protein (GlnBP) induced by GdnHCl and the effect of the binding of glutamine (Gln) on these processes. To this end, GdnHCl-induced unfolding of GlnBP alone and its GlnBP-Gln complex was studied by protein intrinsic fluorescence, ANS emission fluorescence, and far- and near-UV circular dichroism spectroscopy. The obtained spectroscopic data were interpreted taking into the account the peculiarities of protein three-dimensional structure. In particular, the fact that formation of a complex of GlnBP and Gln, which essentially changes the global structure of protein, affects only insignificantly the microenvironments of tryptophan residues elucidates the similarity of the emission spectra of GlnBP and the GlnBP-Gln complex, and the existence of quenching groups near tyrosine residues and an effective nonradiative Tyr --> Trp and/or Tyr --> Tyr --> Trp energy transfer provide an explanation for the negligibly small contribution of tyrosine to the bulk fluorescence of the native protein and for its increase in protein unfolding. The use of the parametric presentation of fluorescence data showed that both GlnBP unfolding and GlnBP-Gln unfolding are three-step processes (N --> I(1) --> I(2) --> U), though in the case of the GlnBP-Gln complex these stages essentially overlap. Despite the complex character, GlnBP unfolding is completely reversible. The dramatic shift of the N --> I(1) process to higher GdnHCl concentrations for the GlnBP-Gln complex in comparison with GlnBP was shown.     

Crystal structure of Escherichia coli methionyl-tRNA synthetase at 2.5 Å resolution

Native methionyl-tRNA synthetase from Escherichia coli (a dimer of molecular weight 172,000) can be converted by mild proteolysis into a well-defined monomeric fragment of molecular weight 64,000. This fragment retains full specificity towards methionine and tRNA^Met, and has unimpaired activity in both the activation and aminoacylation reactions.This paper describes the structure of the active fragment, as determined by an X-ray crystallographic study at 2.5 Å resolution using five heavy-atom derivatives. The elongated molecule (90 Å × 52 Å × 44 Å) contains several α-helices, which account for 43% of the residues. Three domains can be distinguished in the structure: (1) a central core beginning at the N-terminus, consisting of a five-stranded parallel pleated sheet with α-helices connecting the β-strands; (2) a second domain with less-ordered structure, inserted between the third and fourth strand of the central sheet; (3) a C-terminal domain, beginning after the fifth parallel strand, very rich in α-helices.These three domains are organized in a biglobular structure; one globule contains the first and the second domain (N-terminal globule), the other the third domain. The two globules, linked together by a single chain, are separated by a large cleft.The most salient feature of the structure is the presence, in the N-terminal domain, of a “nucleotide binding fold” similar to that first observed in dehydrogenases. This makes methionyl-tRNA synthetase, and possibly all aminoacyl-tRNA synthetases, a new member of this family of nucleotide binding proteins possessing the characteristic “Rossmann fold”.     

Three-dimensional structure of actinoxanthin. IV. A 2.5-Å resolution

The protein actinoxanthin (isolated from Actinomyces globisporus—molecular weight, 10,300; 107 amino acid residues) crystallizes in space group P2₁2₁2₁ with cell dimensions: a = 30.9 Å, b = 48.8 Å, c = 64.1 Å, and Z = 4. The three-dimensional structure of actinoxanthin was determined by the x-ray multiple isomorphous replacement method at 2.5-Å resolution. The molecule is kidney-shaped and has a well-defined cavity. Its characteristic features are the absence of α-helices and the presence of enhanced content of antiparallel β-structure (～55%). A cylinder-shaped formation of seven antiparallel β-strands comprises the main part of the protein structure. The established β-supersecondary structure is characterized by a three-dimensional topology similar to that of immunoglobulin domains, superoxide dismutase subunits, and azurin and plastocyanin proteins.     

Crystal structure of LeuD from Methanococcus jannaschii

3-Isopropylmalate/citramalate (IPM) isomerase catalyzes the second step in the leucine biosynthesis pathway. IPM isomerase from Methanococcus jannaschii is a complex protein consisting of a large (MjLeuC) and a small subunit (MjLeuD). It has broad substrate specificity, unlike other bacterial IPM isomerases. In order to understand the reasons for this broad substrate specificity, we determined the crystal structure of MjLeuD at a resolution of 2.0 Å. The asymmetric unit contained 6 molecules of LeuD, including three homodimers. The overall structure had a β/β/α sandwich-fold consisting of 8 α-helices and 7 β-strands. The C-terminal helix, which is important in homodimer formation, showed conformational differences between two homodimer forms of MjLeuD. In addition, we identified a hydrophobic residue (Val28) near the substrate recognition region that may explain the broad substrate specificity of IPM isomerase. Therefore, we suggest that LeuD proteins can be divided into 2 subfamilies, LeuD subfamilies 1 and 2, which show differences in overall structure and in the substrate recognition region.     

An empirical energy function for threading protein sequence through the folding motif

In this paper we present a new residue contact potantial derived by statistical analysis of protein crystal structures. This gives mean hydrophobic and pairwise contact energies as a function of residue type and distance interval. To test the accuracy of this potential we generate model structures by “threading” different sequences through backbone folding motifs found in the structural data base. We find that conformational energies calculated by summing contact potentials show perfect specificity in matching the correct sequences with each globular folding motif in a 161-protcin data set. They also identify correct models with the core folding motifs of heme-rythrin and immunoglobulin McPC603 V₁-do- main, among millions of alternatives possible when we align subsequences with α-helices and β-strands, and allow for variation in the lengths of intervening loops. We suggest that contact potentials reflect important constraints on nonbonded interaction in native proteins, and that “threading” may be useful for structure prediction by recognition of folding motif. © 1993 Wiley-Liss, Inc.     

Twisted hyperboloid (strophoid) as a model of β-barrels in proteins

Using a least-squares fitting procedure, polypeptide backbones of one parallel and seven antiparallel β-barrels were approximated with various curved surfaces. Although the hyperboloid gave better approximations to all the β-barrel backbones than the ellipsoid, elliptical cylinder or catenoid, the best approximations were obtained with a novel surface, a twisted hyperboloid (strophoid). The root-mean-square errors between individual β-barrels and the fitted strophoid surfaces ranged from 0.75 Å to 1.64 Å. The parameters which determine the strophoid surface allow groups of β-barrel shapes to be defined according to their barrel twists (i.e. angles subtended by directions of the long axis of cross-section at the top and the bottom of the barrel), course of elliptical cross-sections (either monotonically increasing along the barrel axis, as in cones, or having a middle “waist”, as in hyperboloids), and types of backbone curvatures (either convex or concave). The curvatures at individual points of strophoid surface are local, variable quantities related to the local helicity (coil) of the polypeptide backbone, in contrast to values of β-sheet twist (i.e. dihedral angles subtended by adjacent β-strands) known to be virtually identical in all the β-sheets. The variability found in parameters such as barrel shapes and curvatures suggests that simple models (isotropically stressed surfaces, principle of minimal surface tension) proposed in the past to account for β-barrel shapes are not sufficient. Rather, the complex nature of best-fit theoretical surfaces points to an important role played by a local variability of the forces involved.     

Novel structure of the conserved gram-negative lipopolysaccharide transport protein A and mutagenesis analysis

Lipopolysaccharide (LPS) transport protein A (LptA) is an essential periplasmic localized transport protein that has been implicated together with MsbA, LptB, and the Imp/RlpB complex in LPS transport from the inner membrane to the outer membrane, thereby contributing to building the cell envelope in Gram-negative bacteria and maintaining its integrity. Here we present the first crystal structures of processed Escherichia coli LptA in two crystal forms, one with two molecules in the asymmetric unit and the other with eight. In both crystal forms, severe anisotropic diffraction was corrected, which facilitated model building and structural refinement. The eight-molecule form of LptA is induced when LPS or Ra-LPS (a rough chemotype of LPS) is included during crystallization. The unique LptA structure represents a novel fold, consisting of 16 consecutive antiparallel β-strands, folded to resemble a slightly twisted β-jellyroll. Each LptA molecule interacts with an adjacent LptA molecule in a head-to-tail fashion to resemble long fibers. Site-directed mutagenesis of conserved residues located within a cluster that delineate the N-terminal β-strands of LptA does not impair the function of the protein, although their overexpression appears more detrimental to LPS transport compared with wild-type LptA. Moreover, altered expression of both wild-type and mutated proteins interfered with normal LPS transport as witnessed by the production of an anomalous form of LPS. Structural analysis suggests that head-to-tail stacking of LptA molecules could be destabilized by the mutation, thereby potentially contributing to impair LPS transport.     

Prediction of the structure of the replication initiator protein DnaA

The secondary structure of DnaA protein and its interaction with DNA and ribonucleotides has been predicted using biochemical, biophysical techniques, and prediction methods based on multiple-sequence alignment and neural networks. The core of all proteins from the DnaA family consists of an “open twisted α/β structure,” containing five α-helices alternating with five β-strands. In our proposed structural model the interior of the core is formed by a parallel β-sheet, whereas the α-helices are arranged on the surface of the core. The ATP-binding motif is located within the core, in a loop region following the first β-strand. The N-terminal domain (80 aa) is composed of two α-helices, the first of which contains a potential leucine zipper motif for mediating protein-protein interaction, followed by a β-strand and an additional α-helix. The N-terminal domain and the α/β core region of DnaA are connected by a variable loop (45–70 aa); major parts of the loop region can be deleted without loss of protein activity. The C-terminal DNA-binding domain (94 aa) is mostly α-helical and contains a potential helix-loop-helix motif. DnaA protein does not dimerize in solution; instead, the two longest C-terminal α-helices could interact with each other, forming an internal “coiled coil” and exposing highly basic residues of a small loop region on the surface, probably responsible for DNA backbone contacts. © 1997 Wiley-Liss Inc.     

TonB-dependent receptors—structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel β-barrel traverses the outer membrane and adjacent β-strands are connected by extracellular loops and periplasmic turns. Located inside the β-barrel is the plug domain, composed primarily of a mixed four-stranded β-sheet and a series of interspersed α-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

Proton Nuclear Magnetic Resonance Studies on Huwentoxin-I from the Venom of the Spider Selenocosmia huwena: 2. Three-Dimensional Structure in Solution

The three-dimensional structure in aqueous solution of native huwentoxin-I, a neurotoxin from the venom of the spider Selenocosmia huwena, has been determined from two-dimensional ¹H NMR data recorded at 500 and 600 MHz. Structural constraints consisting of interproton distances inferred from NOEs and dihedral angles from spin–spin coupling constants were used as input for distance geometry calculation with the program XPLOR 3.1. The best 10 structures have NOE violations <0.3 Å, dihedral violations <2°, and pairwise root-mean-square differences of 1.08 (±0.20) Å over backbone atoms (N, C^α;, C). The molecule adopts a compact structure consisting of a small triple-stranded antiparallel β-sheet and five β-turns. A small hydrophobic patch consisting of Phe 6, Trp 28, and Trp 31 is located on one side of the molecule. All six lysine residues are distributed on the molecular surface. The three disulfidc bridges are buried within the molecule. The structure contains an “inhibitor cystine knot motif” which is adopted by several other small proteins, such as ω-conotoxin, agatoxin IVA, and gurmarin.     

Three-Dimensional Structure of Selenocosmia huwena Lectin-I (SHL-I) from the Venom of the Spider Selenocosmia huwena by 2D-NMR

The three-dimensional structure of native SHL-I, a lectin from the venom of the Chinese bird spider Selenocosmia huwena, has been determined from two-dimensional ¹H NMR spectroscopy recorded at 500 and 600 MHz. The best 10 structures have NOE violation <0.3 Å, dihedral violation <2 deg, and average root-mean-square differences of 0.85 + 0.06 Å over backbone atoms. The structure consists of a three-stranded antiparallel β-sheet and three turns. The three disulfide bridges and three-stranded antiparallel β-sheet form a inhibitor cystine knot motif which is adopted by several other small proteins, such as huwentoxin-I, ω-conotoxin, and gurmarin. The C-terminal fragment from Leu28 to Trp32 adopts two sets of conformations corresponding to the cis and trans conformations of Pro31. The structure of SHL-I also has high similarity with that of the N-terminus of hevein, a lectin from rubber-tree latex.     

Two conformations of crystalline adenylate kinase.

Pig muscle adenylate kinase (EC2.7.4.3) can exist in three crystal forms, which are interconvertible. For crystal form A the enzyme structure is known in atomic detail. We report the X-ray diffraction analysis of crystal form B at 4.7 Å resolution and a comparison with the A form. During the transition from A to B the packing arrangement of the molecules changes slightly. Moreover, the individual molecule undergoes an appreciable conformational change: by displacing a chain segment of seven residues and two adjacent α-helices a hydrophobic pocket is opened deep in the cleft near the centre of the molecule. Concomitantly the β-pleated sheet is enlarged by about four hydrogen bonds in the B form. Several lines of evidence indicate that the observed conformational change is an intrinsic property of the molecule and is not induced by crystal packing forces.     

NMR assignments of the GacS histidine-kinase periplasmic detection domain from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Pseudomonas aeruginosa is a highly adaptable opportunistic pathogen. It can infect vulnerable patients such as those with cystic fibrosis or hospitalized in intensive care units where it is responsible for both acute and chronic infection. The switch between these infections is controlled by a complex regulatory system involving the central GacS/GacA two-component system that activates the production of two small non-coding RNAs. GacS is a histidine kinase harboring one periplasmic detection domain, two inner-membrane helices and three H1/D1/H2 cytoplasmic domains. By detecting a yet unknown signal, the GacS histidine-kinase periplasmic detection domain (GacSp) is predicted to play a key role in activating the GacS/GacA pathway. Here, we present the chemical shift assignment of 96 % of backbone atoms (HN, N, C, Cα, Cβ and Hα), 88 % aliphatic hydrogen atoms and 90 % of aliphatic carbon atoms of this domain. The NMR-chemical shift data, on the basis of Talos server secondary structure predictions, reveal that GacSp consists of 3 β-strands, 3 α-helices and a major loop devoid of secondary structures.     

Structure of trichosanthin at 1.88 Å resolution

Trichosanthin (TCS) is one of the single chain ribosome-inactivating proteins (RIPs). The crystals of the orthorhombic form of trichosanthin have been obtained from a citrate buffer (pH 5.4) with KC1 as the precipitant. The crystal belongs to the space group P2₁2₁2₁ with a = 38.31, b = 76.22, c = 79.21 Å. The structure was solved by molecular replacement method and refined using the programs XPLOR and PROLSQ to an R-factor of 0.191 for the reflections within the 6–1.88 Å resolution range. The bond length and bond angle in the protein molecule have root-mean-square deviations from ideal value of 0.013 Å and 3.3°, respectively. The refined model includes 247 residues and 197 water molecules. The TCS molecule consists of two structural domains. The large domain contains six α-helices, a six stranded sheet, and an antiparallel β-sheet. The small domain has a largest α-helix, which shows a distinct bend. The possible active site of the molecule located on the cleft between two domains was proposed. In the active site Arg-163 and Glu-160, Glu-189 and Arg-122 form two ion pairs, Glu-189 and Gln-156 are hydrogen bonded to each other. Three water molecules are bonded to the residues in the active site region. The structures of TCS molecule and ricin A-chain (RTA) superimpose quite well, showing that the structures of the two protein molecules are homologous. Comparison of the structures of the TCS molecule in this orthorhombic crystal with that in the monoclinic crystal indicates that there are no essential differences of the structures between the two protein crystals. © 1994 Wiley-Liss, Inc.     

Do parallel β-strands have dipole moments? An ab initio molecular-orbital-direct reaction field study

Recently, it was suggested that parallel β-sheets have a significant dipole moment, in contrast to antiparallel sheets. Ab initio molecular-orbital (MO) calculations on parallel and antiparallel β-strands of tetra(Gly) show that they have very similar charge distributions. Interaction energies between two and three strands of tetra(Gly), obtained using the direct reaction field Hamiltonian, show that a particular choice of point charges is probably not crucial for calculating interactions within β-sheets, but that it might be for calculating interactions between these sheets and other parts of a protein, in particular, α-helices. The point-charge representation of our MO-SCF results will probably reduce the hazard of introducing artefacts in electrostatic calculations of protein conformational energies, provided the short-range interactions are treated in a more realistic way, i.e., such that intra- and interchain induction effects are included.     

Predominant end-products of prophage Mu DNA transposition during the lytic cycle are replicon fusions

The structure of l-arabinose-binding protein (M_r 33, 100), an essential component of the osmotic shock-sensitive, high-affinity l-arabinose transport system in Escherichia coli, has been determined at 2.4 Å resolution. The phases were solved by the method of multiple isomorphous replacement, using four derivatives, p-chloromercuribenzenesulfonate and CdI₂ (data to 2.4 Å resolution), and p-chloromercurinitrophenol and (NH₄)₂PtCl₄ to 3.5 Å resolution. A final mean figure of merit of 0.65 was obtained for 9628 reflections.With the aid of the amino acid sequence determined by Hogg &; Hermodson (1977), a complete model of the protein molecule has been determined using initially an optical comparator. The entire model was subsequently examined in detail using a computer graphic system.The protein molecule is ellipsoidal (axial ratio of 2:1), and consists of two globular domains (designated P and Q). Each domain is made from two separate polypeptide chain segments. Despite the discontinuity in the folding, the arrangements of the secondary structure in the two domains are very similar. Both domains contain a six-stranded parallel β-sheet (with the exception of the sixth anti-parallel strand in the Q domain) flanked by two α-helices on either side. The packing topology is α/β. A C-terminal helix is shared by both domains.The two domains show significant conformational similarity but lack sequence homology. A comparison of the two domains revealed that of the 139 α-carbons in the P domain and 152 in the Q domain, 92 were found to be equivalent with a root-mean-square distance of 2.6 Å.The cleft formed by the packing of the two domains is predominantly lined with hydrophilic residues. The sugar-binding site is located in this cleft.     

Solution Structure of the Lipoyl Domain of the 2-Oxoglutarate Dehydrogenase Complex fromAzotobacter vinelandii

The three-dimensional solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex fromAzotobacter vinelandiihas been determined from nuclear magnetic resonance data by using distance geometry and dynamical simulated annealing refinement. The structure determination is based on a total of 580 experimentally derived distance constraints and 65 dihedral angle constraints. The solution structure is represented by an ensemble of 25 structures with an average root-mean-square deviation between the individual structures of the ensemble and the mean coordinates of 0.71 Å for backbone atoms and 1.08 Å for all heavy atoms. The overall fold of the lipoyl domain is that of a β-barrel-sandwich hybrid. It consists of two almost parallel four-stranded anti-parallel β-sheets formed around a well-defined hydrophobic core, with a central position of the single tryptophan 21. The lipoylation site, lysine 42, is found in a β-turn at the far end of one of the sheets, and is close in space to a solvent-exposed loop comprising residues 7 to 15. The lipoyl domain displays a remarkable internal symmetry that projects one β-sheet onto the other β-sheet after rotation of approximately 180° about a 2-fold rotational symmetry axis. There is close structural similarity between the structure of this 2-oxoglutarate dehydrogenase complex lipoyl domain and the structures of the lipoyl domains of pyruvate dehydrogenase complexes fromBacillus stearothermophilusandEscherichia coli, and conformational differences occur primarily in a solvent-exposed loop close in space to the lipoylation site. The lipoyl domain structure is discussed in relation to the process of molecular recognition of lipoyl domains by their parent 2-oxo acid dehydrogenase.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Structure analysis and molecular model of the selenoenzyme glutathione peroxidase at 2.8 Å resolution

The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole (M_r = 84,000), has been determined at 2.8 Å resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar.The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of $\text{r ≈ 19}\text{A}\text{?}$. A tentative sequence corresponding to a partially refined model (R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S₂ revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold.The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).