Antiparallel Beta-barrel Research Articles

Three-dimensional structure of 6-pyruvoyl tetrahydropterin synthase, an enzyme involved in tetrahydrobiopterin biosynthesis.

The crystal structure of rat liver 6-pyruvoyl tetrahydropterin synthase has been solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.4% at 2.3 A resolution. 6-Pyruvoyl tetrahydrobiopterin synthase catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. The functional enzyme is a hexamer of identical subunits. The 6-pyruvoyl tetrahydropterin synthase monomer folds into a sequential, four-stranded, antiparallel beta-sheet with a 25 residue, helix-containing insertion between strands 1 and 2 at the bottom of the molecule, and a segment between strands 2 and 3 forming a pair of antiparallel helices, layered on one side of the beta-sheet. Three 6-pyruvoyl tetrahydropterin synthase monomers form an unusual 12-stranded antiparallel beta-barrel by tight association between the N- and C-terminal beta-strands of two adjacent subunits. The barrel encloses a highly basic pore of 6-12 A diameter. Two trimers associate in a head-to-head fashion to form the active enzyme complex. The substrate-binding site is located close to the trimer-trimer interface and comprises residues from three monomers: A, A' and B. A metal-binding site in the substrate-binding pocket is formed by the three histidine residues 23, 48 and 50 from one 6-pyruvoyl tetrahydropterin synthase subunit. Close to the metal, but apparently not liganding it, are residues Cys42, Glu133 (both from A) and His89 (from B), which might serve as proton donors and acceptors during catalysis.

Plastocyanin: structural and functional analysis.

Plastocyanin is one of the best characterized of the photosynthetic electron transfer proteins. Since the determination of the structure of poplar plastocyanin in 1978, the structure of algal (Scenedesmus, Enteromorpha, Chlamydomonas) and plant (French bean) plastocyanins has been determined either by crystallographic or NMR methods, and the poplar structure has been refined to 1.33 A resolution. Despite the sequence divergence among plastocyanins of algae and vascular plants (e.g., 62% sequence identity between the Chlamydomonas and poplar proteins), the three-dimensional structures are remarkably conserved (e.g., 0.76 A rms deviation in the C alpha positions between the Chlamydomonas and poplar proteins). Structural features include a distorted tetrahedral copper binding site at one end of an eight-stranded antiparallel beta-barrel, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and the negative and hydrophobic patches are proposed to be involved in recognition of physiological reaction partners. Chemical modification, cross-linking, and site-directed mutagenesis experiments have confirmed the importance of the negative and hydrophobic patches in binding interactions with cytochrome f and Photosystem I, and validated the model of two functionally significant electron transfer paths in plastocyanin. One putative electron transfer path is relatively short (approximately 4 A) and involves the solvent-exposed copper ligand His-87 in the hydrophobic patch, while the other is more lengthy (approximately 12-15 A) and involves the nearly conserved residue Tyr-83 in the negative patch.

Crystal structure of apo-avidin from hen egg-white

The three-dimensional structure of hen egg-white apo-avidin, crystallized in a tetragonal crystal form, has been refined to a crystallographic R-factor of 0.164 (for the 6390 observed reflections in the 10.0 to 2.8 A resolution range). As in the case of holo-avidin, from which starting atomic co-ordinates were derived, the functional tetramer shows 2-pseudo 22 molecular symmetry. Each promoter is organized in an eight-stranded antiparallel orthogonal beta-barrel, with extended loop regions, which define the biotin binding pocket in the protomer core. In the absence of biotin the binding site is only partly occupied by water molecules. The structure of the binding site residues, as observed in apo-avidin, is highly complementary to that of the incoming biotin molecule, accounting for prompt and specific recognition. A crystal lattice contact may play a role in stabilizing the conformation of one protein loop, part of the biotin-binding pocket.

Evidence that facilitative glucose transporters may fold as beta-barrels.

A widely accepted model for the structure of the facilitative glucose transporters (GLUTs) predicts that they form 12 transmembrane alpha-helices and that the highly conserved sequence Ile-386-Ala-405 in GLUT1 is intracellular. We raised a polyclonal antibody against a synthetic peptide encompassing this conserved sequence and found that antibody treatment increased 2-deoxy-D-glucose (DOG) uptake in Xe-nopus oocytes expressing GLUT1, GLUT2, or GLUT4 only when applied to the extracellular side. This effect was dose dependent and was specifically blocked by competition with the peptide Ile-386-Ala-405; it was due to a decrease in the Km for the transport of DOG. To ascertain GLUT orientation, we raised anti-peptide antibodies against the last 21 and 25 C-terminal amino acids of GLUT1 and GLUT4, respectively, which were previously shown to be intracellular. These antibodies increased DOG uptake when injected into oocytes expressing GLUT1 and GLUT4, but not when added extracellularly. Prompted by the noted discrepancy, we found sequence similarity between GLUTs and porins, two of which are known from crystallography to form 16-stranded transmembrane antiparallel beta-barrels. Analysis of the hydrophobicity, amphiphilicity, and turn propensity of GLUT1 leads us to propose that GLUTs fold as porin-like transmembrane beta-barrels. This model is consistent with the results of the present antibody studies and also with previously published experimental evidence inconsistent with the 12-helix model.

The 1.5-.ANG. crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii

The crystal structure of plastocyanin from the green alga Chlamydomonas reinhardtii has been determined at 1.5-A resolution with a crystallographic R factor of 16.8%. Plastocyanin is a small (98 amino acids), blue copper-binding protein that catalyzes the transfer of electrons in oxygenic photosynthesis from cytochrome f in the quinol oxidase complex to P700+ in photosystem I. Chlamydomonas reinhardtii plastocyanin is an eight-stranded, antiparallel beta-barrel with a single copper atom coordinated in quasitetrahedral geometry by two imidazole nitrogens (from His-37 and His-87), a cysteine sulfur (from Cys-84), and a methionine sulfur (from Met-92). The molecule contains a region of negative charge surrounding Tyr-83 (the putative distant site of electron transfer) and an exclusively hydrophobic region surrounding His-87; these regions are thought to be involved in the recognition of reaction partners for the purpose of directing electron transfer. Chlamydomonas reinhardtii plastocyanin is similar to the other plastocyanins of known structure, particularly the green algal plastocyanins from Enteromorpha prolifera and Scenedesmus obliquus. A potential "through-bond" path of electron transfer has been identified in the protein that involves the side chain of Tyr-83, the main-chain atoms between residues 83 and 84, the side chain of Cys-84, the copper atom, and the side chain of His-87.

Structure in solution of the major cold-shock protein from Bacillus subtilis.

The cold-shock domain (CSD) is found in many eukaryotic transcriptional factors and is responsible for the specific binding to DNA of a cis-element called the Y-box. The same domain exists in the sequence of the Xenopus RNA-binding proteins FRG Y1 and FRG Y2 (refs 1, 3). The major cold-shock proteins of Escherichia coli (CS7.4) and B. subtilis (CspB) have sequences that are more than 40 per cent identical to the cold-shock domain. We present here the three-dimensional structure of CspB determined by nuclear magnetic resonance spectroscopy. The 67-residue protein consists of an antiparallel five-stranded beta-barrel with strands connected by turns and loops. The structure resembles that of staphylococcal nuclease and the gene-5 single-stranded-DNA-binding protein. A three-stranded beta-sheet, which contains the conserved RNA-binding motif RNP1 as well as a motif similar to RNP2 in two neighbouring antiparallel beta-strands, has basic and aromatic residues at its surface which could serve as a binding site for single-stranded DNA. CspB binds to single-stranded DNA in gel retardation experiments.

Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein.

The cold-shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature. It leads to the increased production of the major cold-shock proteins, CS7.4 and CspB, respectively. CS7.4 is a transcriptional activator of two genes. CS7.4 and CspB share 43 per cent sequence identity with the nucleic acid-binding domain of the eukaryotic gene-regulatory Y-box factors. This cold-shock domain is conserved from bacteria to man and contains the RNA-binding RNP1 sequence motif. As a prototype of the cold-shock domain, the structure of CspB has been determined here from two crystal forms. In both, CspB is present as an antiparallel five-stranded beta-barrel. Three consecutive beta-strands, the central one containing the RNP1 motif, create a surface rich in aromatic and basic residues that are presumably involved in nucleic acid binding. Preferential binding of CspB to single-stranded DNA is observed in gel retardation experiments.

Functional interactions between K+ pore residues located in different subunits

The aqueous pore (P-region) of homotetrameric voltage-gated K+ channels has been modeled as a radially symmetrical eight-stranded antiparallel beta-barrel to which each of the four subunits contributes equally. This model has hydrogen bonding between residues located on adjacent subunits and predicts that subunit interactions might have functional consequences. Previously we have used point mutations and an electrophysiological assay to detect functional interactions between a pair of residues at positions 369 and 374 in the P-region, but we could not distinguish between intra- and intersubunit interactions. In the present paper, we present evidence for interaction across subunit boundaries after co-injecting two cRNAs encoding subunits differing from each other at either position 369 or 374. Comparison of the phenotypes of homo- and heterotetrameric channels suggests that pore residues residing in adjacent subunits form a closely packed structure which determines both ion conductance and stability of the open state of the channel. Our results are consistent with a structure in which pore residues 369 and 374 are located in close proximity on adjacent antiparallel strands to allow both intra- and intersubunit interactions.

Protein motif by computer: the perfect Greek key jellyroll designer.

A program that generates amino acid sequences that are compatible with the Greek key protein motif is presented. Using statistical data derived from the structures of molecules from the protein databank, the novel algorithm generates amino acid sequences compatible with an 8-stranded perfect Greek key jellyroll motif. In this motif, all hydrogen bonds present in the theoretical originating beta-hairpin stay in register as the whole 8-stranded domain folds at once in an anticlockwise swirl. Eight residues are generated per strand and 32 residues per sheet making 64 residues in the antiparallel beta-barrel. The seven loops between beta-strands contain an additional 27 residues. All recognized features of beta-sheets and beta-strands, such as alternating hydrophobic, hydrophilic residues with hydrophobics on the narrow-hydrogen-bond-pair, concave side of the theoretical originating beta-ribbon; sheet twist, strand twist, side chain rotation about the strands; the theories of side chain packing between the sheets; an average 30 degrees rotation between beta-sheets; the theoretical anticomplementary patch residues of each sheet; and the anticomplementary, isotropically stressed hyperbolic parabloid shape of each sheet, are taken into account in the program. The sequences of the loops between strands are designed by turn type and strand twist is considered in the design of the motif's single beta-hairpin turn. Secondary structure parameters and between-strand amino acid pair correlations also figure importantly in the novel algorithm.

Crystal structure at 1.7 A of the bovine papillomavirus-1 E2 DNA-binding domain bound to its DNA target.

The dominant transcriptional regulator of the papillomaviruses, E2, binds to its specific DNA target through a previously unobserved dimeric antiparallel beta-barrel. The DNA is severely but smoothly bent over the barrel by the interaction of successive major grooves with a pair of symmetrically disposed alpha-helices. The specific interface is an 'interwoven' network of interactions where the identifying base pairs of the target contact more than one amino-acid side chain and the discriminating amino acids interact with more than one base pair.

Crystal structures explain functional properties of two E. coli porins.

Porins form aqueous channels that aid the diffusion of small hydrophilic molecules across the outer membrane of Gram-negative bacteria. The crystal structures of matrix porin and phosphoporin both reveal trimers of identical subunits, each subunit consisting of a 16-stranded anti-parallel beta-barrel containing a pore. A long loop inside the barrel contributes to a constriction of the channel where the charge distribution affects ion selectivity. The structures explain at the molecular level functional characteristics and their alterations by known mutations.

Sequential 1H and 15N NMR assignments and secondary structure of a recombinant anti-digoxin antibody VL domain.

A uniformly 15N-labeled recombinant light-chain variable (VL) domain from the anti-digoxin antibody 26-10 has been investigated by heteronuclear two-dimensional (2D) and three-dimensional (3D) NMR spectroscopy. Complementary homonuclear 2D NMR studies of the unlabeled VL domain were also performed. Sequence-specific assignments for 97% of the main-chain and 70% of the side-chain proton resonances have been obtained. Patterns of nuclear Overhauser effects observed in 2D NOESY, 3D NOESY-HSQC, and 3D NOESY-TOCSY-HSQC spectra afford a detailed characterization of the VL domain secondary structure in solution. The observed secondary structure--a nine-stranded antiparallel beta-barrel--corresponds to that observed crystallographically for VL domains involved in quaternary associations. The locations of slowly exchanging amide protons have been discerned from a 2D TOCSY spectrum recorded after dissolving the protein in 2H2O. Strands B, C, E, and F are found to be particularly stable. The possible consequences of these results for domain-domain interactions are discussed.

Identification of candidate sequences that determine virulence in Coxsackievirus B4.

We have previously shown that a major determinant of virulence for coxsackievirus B4 mapped to the 5' end of the viral genome. Comparison of the corresponding cDNA sequences of a virulent and a non-virulent virus has allowed the identification of candidate determinants of virulence in the 5' untranslated region and the capsid proteins VP1, VP2 and VP4. Thirteen nucleotide substitutions were observed in a region spanning 3298 nucleotides. Four mutations were detected in the non-coding region. Of the remaining nine mutations, four were silent while five resulted in amino acid substitutions in VP1, VP2 and VP4. The amino acid substitutions in the virulent virus were analyzed in relation to the three-dimensional structures of the capsid proteins of poliovirus. Two substitutions mapped to the amino termini of VP1 and VP4. Of the two substitutions observed in VP2, one mapped to the large loop that connects beta strand E with the radial helix on the back surface of the eight-stranded antiparallel beta barrel while the other mapped to beta strand G. One amino acid substitution in VP1 mapped to the loop connecting beta strands D and E at a site close to a major determinant of attenuation in poliovirus type 2.

Three-dimensional structure of Theiler virus.

Theiler murine encephalomyelitis virus strains are categorized into two groups, a neurovirulent group that rapidly kills the host, and a demyelinating group that causes a generally nonlethal infection of motor neurons followed by a persistent infection of the white matter with demyelinating lesions similar to those found in multiple sclerosis. The three-dimensional structure of the DA strain, a member of the demyelinating group, has been determined at 2.8 A resolution. As in other picornaviruses, the icosahedral capsid is formed by the packing of wedge-shaped eight-stranded antiparallel beta barrels. The surface of Theiler virus has large star-shaped plateaus at the fivefold axes and broad depressions spanning the twofold axes. Several unusual structural features are clustered near one edge of the depression. These include two finger-like loops projecting from the surface (one formed by residues 78-85 of VP1, and the other formed by residues 56-65 of VP3) and a third loop containing three cysteines (residues 87, 89, and 91 of VP3), which appear to be covalently modified. Most of the sequence differences between the demyelinating and neurovirulent groups that could play a role in determining pathogenesis map to the surface of the star-shaped plateau. The distribution of these sequence differences on the surface of the virion is consistent with models in which the differences in the pathogenesis of the two groups of Theiler viruses are the result of differences in immunological or receptor-mediated recognition processes.

Three-dimensional solution structure of apo-neocarzinostatin from Streptomyces carzinostaticus determined by NMR spectroscopy.

The three-dimensional solution structure of apo-neocarzinostatin has been resolved from nuclear magnetic resonance spectroscopy data. Up to 1034 constraints were used to generate an initial set of 45 structures using a distance geometry algorithm (DSPACE). From this set, ten structures were subjected to refinement by restrained energy minimization and molecular dynamics. The average atomic root mean square deviations between the final ten structures and the mean structure obtained by averaging their coordinates run from 0.085 nm for the best defined beta-sheet regions of the protein to 0.227 nm for the side chains of the most flexible loops. The solution structure of apo-neocarzinostatin is closely similar to that of the related proteins, macromomycin and actinoxanthin. It contains a seven-stranded antiparallel beta-barrel which forms, together with two external loops, a deep cavity that is the chromophore binding site. It is noteworthy that aromatic side chains extend into the binding cleft. They may be responsible for the stabilization of the holo-protein complex and for the chromophore specificity within the antitumoral family.

An SS1-SS2 beta-barrel structure for the voltage-activated potassium channel.

To examine the feasibility of a beta structure for the pore-lining region of the voltage-gated potassium channel, we have characterized a family of 12 antiparallel beta-barrels. Each is comprised of four identical pairs of beta-strands organized with approximate 4-fold symmetry about a channel axis. The C- and N-termini of the beta-strand pairs are assumed to be at the extracellular end of the channel, and each pair is connected by a hairpin turn at the intracellular end of the channel. The models differ in the residues located in the hairpin turn and in the orientation of the two strands of each pair in the barrel, i.e. whether the C-terminus of a pair is clockwise (CW) or counterclockwise (CCW) from the N-terminus when the channel is viewed from outside the cell. Following known structure precedents and potential energy predictions, the barrel is assumed to be right-twisting in all cases. All models have crowded layers of inward-projecting aromatic side-chains near the center of the channel which could regulate channel selectivity. The models with an odd number of amino acids in the hairpin turn have the advantage of predicting that F433 points into the barrel, but the disadvantage that V438 does not. Of these models, two of the models are most consistent with the external tetraethylammonium (TEA) block data, and of those, one (T439 CCW 3:5) is most consistent with the internal TEA block data.

Structure of the IIA domain of the glucose permease of Bacillus subtilis at 2.2-.ANG. resolution

The crystal structure of the IIA domain of the glucose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) from Bacillus subtilis has been determined at 2.2-A resolution. Refinement of the structure is in progress, and the current R-factor is 0.201 (R = sigma h parallel Fo magnitude of - Fc parallel/sigma h magnitude of Fo, where magnitude of Fo and magnitude of Fc are the observed and calculated structure factor amplitudes, respectively) for data between 6.0- and 2.2-A resolution for which F greater than or equal to 2 sigma (F). This is an antiparallel beta-barrel structure that incorporates "Greek key" and "jellyroll" topological motifs. A shallow depression is formed at the active site by part of the beta-sheet and an omega-loop flanking one side of the sheet. His83, the histidyl residue which is the phosphorylation target of HPr and which transfers the phosphoryl group to the IIB domain of the permease, is located at the C-terminus of a beta-strand. The N epsilon atom is partially solvated and also interacts with the N epsilon atom of a second histidyl residue, His68, located at the N-terminus of an adjacent beta-strand, suggesting they share a proton. The geometry of the hydrogen bond is imperfect, though. Electrostatic interactions with other polar groups and van der Waals contacts with the side chains of two flanking phenylalanine residues assure the precise orientation of the imidazole rings. The hydrophobic nature of the surface around the His83-His68 pair may be required for protein-protein recognition by HPr or/and by the IIB domain of the permease. The side chains of two aspartyl residues, Asp31 and Asp87, are oriented toward each other across a narrow groove, about 7 A from the active-site His83, suggesting they may play a role in protein-protein interaction. A model of the phosphorylated form of the molecule is proposed, in which oxygen atoms of the phosphoryl group interact with the side chain of His68 and with the main-chain nitrogen atom of a neighboring residue, Val89. The model, in conjunction with previously reported site-directed mutagenesis experiments, suggests that the phosphorylation of His83 may be accompanied by the protonation of His68. This may be important for the interaction with the IIB domain of the permease and/or play a catalytic role in the phosphoryl transfer from IIA to IIB.

Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy

The protein cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A. Cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation. Cyclophilin may be responsible for mediating this immunosuppressive response. Cyclophilin also catalyses the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates. Here we report the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and the identification, by nuclear magnetic resonance spectroscopy, of the specific binding site for cyclosporin A. Cyclophilin has an eight-stranded antiparallel beta-barrel structure. The prolyl isomerase substrate-binding site is coincident with the cyclosporine-binding site. These results may help to provide a structural basis for rationalizing the immunosuppressive function of the cyclosporin-cyclophilin system and will also be important in the design of improved immunosuppressant drugs.

The quaternary structure of streptavidin in urea

We report on the interactions of urea and guanidinium salts with streptavidin. Gel filtration chromatography in 0, 4, 6, and 7 M urea indicates that the streptavidin tetramer remains intact in urea. Biotin alters the electrophoretic mobility of streptavidin whether or not 6 M urea is present. The intrinsic fluorescence of streptavidin is increased and blue-shifted in 6 M urea. The fluorescence changes indicate the absence of unfolding. A conformational response to urea is possible, but much of the fluorescence change is due to urea binding as a weak biotin analog (Ka approximately 1.3 M-1). The resistance to structural perturbation by urea reflects the structural stability of streptavidin's anti-parallel beta-barrel motif. Unfolding is sluggish in 6 M guanidinium hydrochloride (half-time, approximately 50 days). After guanidinium thiocyanate unfolding, streptavidin can be refolded, but the unfolding and refolding transitions are centered at different concentrations of perturbant. Slow unfolding, with a 15th power dependence on guanidinium thiocyanate concentration, may be partially responsible for the noncoincidence of the unfolding and refolding processes. Nonequilibrium behavior is also seen in 6 M urea, as native streptavidin does not unfold and guanidinium thiocyanate unfolded streptavidin does not refold. Refolding does occur at lower concentrations of urea. Guanidinium thiocyanate only slowly unfolds the biotin-streptavidin complex. In the presence of biotin, unfolded streptavidin does not refold in 6 M guanidinium thiocyanate or in 6 M urea.

Human brain prostaglandin D synthase has been evolutionarily differentiated from lipophilic-ligand carrier proteins.

cDNAs for glutathione-independent prostaglandin D synthase were isolated from cDNA libraries of human brain. The longest cDNA insert was 837 base pairs long and contained a coding region of 570 base pairs corresponding to 190 amino acid residues with a calculated Mr of 21,016. Between two cDNA inserts isolated from the two different libraries, nucleotide substitutions were observed at 16 positions, including conservative amino acid substitutions at 2 positions and nonconservative substitutions at 5 positions, indicating genetic heterogeneity of this enzyme in humans. The computer-assisted homology search revealed that the enzyme is a member of the lipocalin superfamily, comprising secretory hydrophobic molecule transporters, showing the greatest homology (28.8-29.4% identity; 51.3-53.1% similarity) to alpha 1-microglobulin among the members of this superfamily. In a phylogenetic tree of the superfamily, this enzyme, alpha 1-microglobulin, and the gamma chain of the complement component C8 form a cluster separate from the other 14 members. The two distinctive characteristics of glutathione-independent prostaglandin D synthase, as compared to the other members of this superfamily, are its enzymatic properties and its association with membranes that were probably acquired after evolutionary divergence of the two lipocalins. Based on the observed sequence homology, the tertiary structure of the enzyme was deduced to consist of an eight-stranded anti-parallel beta-barrel forming a hydrophobic pocket. Furthermore, the Cys-65 residue in the pocket, which is conserved only in the human and rat enzymes but not in other lipocalins, was considered to be a putative active site of the enzyme.