Crystal structure of Azotobacter cytochrome c5 at 2.5 A resolution

The crystal structure of cytochrome c5 from Azotobacter vinelandii has been solved and refined to an R value of 0.29 at 2.5 A resolution. The structure of the oxidized protein was solved using a monoclinic crystal form. The structure was solved by multiple isomorphous replacements, re-fit to a solvent-leveled multiple isomorphous replacement map, and refined by restrained least squares. The structure reveals monomers associated about the crystallographic 2-fold axis by hydrophobic contacts at the "exposed heme edge". The overall conformation for the monomer is similar to that of Pseudomonas aeruginosa cytochrome c551. However, relative to a common heme conformation, c5 and c551 differ by an average of 6.8 A over 82 alpha-carbon positions and the propionates of c5 are much more exposed to solvent. The shortest heme--heme contact at the "dimer" interface is 6.3 A (Fe to Fe 16.4 A). Alignment of c5 and c551 shows that the two cytochromes, in spite of sequence differences, have remarkably similar charge distributions. A disulfide stacks on a tyrosine between the N- and C-terminal helices.     

Crystal structure of human serum albumin at 2.5 A resolution.

A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.     

Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution

A high-affinity complex formed between G-actin and plasma vitamin D-binding protein (DBP) is believed to form part of a scavenging system in the plasma for removing actin released from damaged cells. In the study presented here, we describe the crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution. The complex contains one molecule of each protein bound together by extensive ionic, polar, and hydrophobic interactions. It includes an ATP and a calcium ion bound to actin, but no evidence of vitamin D metabolites bound to the DBP. Both actin and DBP are multidomain molecules, two major domains in actin and three in DBP. All of these domains contribute to the interaction between the molecules. DBP enfolds the end of the actin molecule, principally in actin subdomain 3 but with additional interactions in actin subdomain 1. This orientation is similar to the binding of profilin to actin, as predicted from previous studies. The more extensive interactions of DBP give an affinity for actin some 3 orders of magnitude higher than that for profilin. The larger "footprint" of DBP on actin also leads to an overlap with the actin-binding site of gelsolin domain I.     

Crystal structure of Aspergillus niger pH 2.5 acid phosphatase at 2. 4 A resolution.

The crystal structure of Aspergillus niger pH 2.5 acid phosphatase (EC 3.1.3.2) has been determined at 2.4 A resolution. In the crystal, two dimers form a tetramer in which the active sites are easily accessible to substrates. The main contacts in the dimer come from the N termini, each lying on the surface of the neighbouring molecule. The monomer consists of two domains, with the active site located at their interface. The active site has a highly conserved catalytic center and a charge distribution, which explains the highly acidic pH optimum and the broad substrate specificity of the enzyme.     

Crystal structure of a protein-toxin alpha 1-purothionin at 2.5A and a comparison with predicted models

Alpha 1-Purothionin (alpha 1-P), a wheatgerm protein and lytic toxin, has a secondary and tertiary structure similar to that of crambin as revealed by CD and NMR studies. alpha 1-P crystallizes in the tetragonal space group 1422 with unit cell dimensions: a = b = 53.59 and c = 69.79 A. X-ray diffraction data have been measured to 2.5 A Bragg spacing. The crystal structure has been determined by molecular replacement methods, using an energy-minimized alpha 1-P model structure derived from crambin (Whitlow and Teeter: Journal of Biomolecular Structure and Dynamics 2:831-848, 1985, Journal of the American Chemical Society 108:7163-7172, 1986). The energy-minimized model gives a slightly cleaner rotation solution and better refinement against the x-ray data than do the crambin or unminimized alpha 1-P structures. The final crystallographic residual with the data in the 10-2.5 A resolution range is 0.216. The refined alpha 1-P structure has a backbone rms difference of 0.74 A from crambin and 0.55 A from the energy-minimized alpha 1-P model. A low resolution NMR model of alpha 1-P calculated from metric matrix distance geometry and restrained molecular dynamics differs from crambin's backbone by 2.3 A rms deviation (Clore et al.: EMBO Journal 5:2729-2735, 1986). Backbone dihedral angles for our predicted model differ from the refined alpha 1-P structure in only one region (at a turn where there is a deletion relative to crambin). The NMR model had differences in four regions.     

Crystal structure of eosinophil cationic protein at 2.4 A resolution

Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.     

Crystal structure of recombinant human interleukin-1 beta at 2.0 A resolution

The crystal structure of recombinant human interleukin-1 beta (IL-1 beta) has been determined at 2.0 A resolution and refined to a crystallographic R-factor of 0.19. Three heavy-atom derivatives were identified and used for multiple isomorphous replacement phasing. Interpretation of the resulting electron density map revealed a structure in which there are 12 antiparallel beta-strands and no alpha-helix. The single 153-residue polypeptide chain is folded into a six-stranded beta-barrel similar in architecture to the Kunitz-type trypsin inhibitor found in soybeans. The molecule displays approximate 3-fold symmetry about the axis of the beta-barrel. Each successive pair of component strands of the barrel brackets an extensive sequence outside the barrel that includes an additional pair of beta-strands and a prominent loop. Together, these three external segments conceal much of the perimeter and one end of the barrel, leaving only the end supporting the chain termini fully exposed. The structure can be used to identify portions of the polypeptide chain that are exposed on the surface of the molecule, some of which must be epitopes recognized by interleukin-1 beta receptors.     

Crystal structure of a sweet tasting protein thaumatin I, at 1.65 A resolution.

The crystal structure of thaumatin I, a potently sweet protein isolated from the fruits of the West African shrub, Thaumatococcus danielli Benth, has been refined at a resolution better than 1.65 A using a combination of energy minimization and stereochemically restrained least-squares methods. The final model consists of all 207 amino acids, 28 alternate amino acid conformers and 236 waters, with a crystallographic R-factor of 0.145 for 19,877 reflections having F > 4 sigma F between 10.0 A and 1.65 A (R = 0.167 for all 24,022 reflections). The model has good stereochemistry, with root-mean-square deviations from ideal values for bond and angle distances of 0.014 A and 0.029 A, respectively. The estimated root-mean-square co-ordinate error is 0.15 A. The current model confirms the previously reported 3.1 A C alpha trace in both main chain connectivity and disulfide topology, including two disulfide bonds, that differed from the earlier reported biochemical determination. The structure contains three domains. The core of the molecule consists of an eleven-stranded, flattened beta-sandwich folded into two Greek key motifs. All beta-strands in this sandwich are antiparallel except the parallel N-terminal and the C-terminal strands. The average hydrogen bond length in this sandwich is 2.89 A, with an angle of 155.1 degrees. Two beta-bulges are found in one of the sheets. The second domain consists of two beta-strands forming a beta-ribbon and connected by an omega-loop, and contains a proline residue in cis conformation. This structural motif folds back against the main sandwich to form a smaller sandwich-like structure. The third domain is a disulfide-rich region stretching away from the sandwich portion of the molecule. It contains one alpha-helix and three short helical fragments. Two of the helical segments are connected by an unusually sharp turn, stabilized by a disulfide bridge. One of the three disulfide bonds in this domain takes on two conformations.     

Crystal structure of schistatin, a disintegrin homodimer from saw-scaled viper (Echis carinatus) at 2.5 A resolution

This is the first structure of a biological homodimer of disintegrin. Disintegrins are a class of small (4-14 kDa) proteins that bind to transmembrane integrins selectively. The present molecule is the first homodimer that has been isolated from the venom of Echis carinatus. The monomeric chain contains 64 amino acid residues. The three-dimensional structure of schistatin has been determined by the multiple isomorphous replacement method. It has been refined to an R-factor of 0.190 using all the data to 2.5 A resolution. The two subunits of the disintegrin homodimer are related by a 2-fold crystallographic symmetry. Thus, the crystallographic asymmetric unit contains a monomer of disintegrin. The monomer folds into an up-down topology with three sets of antiparallel beta-strands. The structure is well ordered with four intramolecular disulfide bonds. the two monomers are firmly linked to each other through two intermolecular disulfide bridges at their N termini together with several other interactions. This structure has corrected the error in the disulfide bond pattern of the two intermolecular disulfide bridges that was reported earlier using chemical methods. Unique sequence and structural features of the schistatin monomers suggest that they have the ability to bind well with both alphaIIb beta3 and alphav beta3 integrins. The N termini anchored two chains of the dimer diverge away at their C termini exposing the Arg-Gly-Asp motif into opposite directions thus enhancing their binding efficiency to integrins. This is one of the unique features of the present disintegrin homodimer and seems to be responsible for the clustering of integrin molecules. The homodimer binds to integrins apparently with a higher affinity than the monomers and also plays a role in the signaling pathway.     

Crystal structure of yeast Cu,Zn superoxide dismutase. Crystallographic refinement at 2.5 A resolution.

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P2(1)2(1)2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded beta-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 beta-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c beta-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 beta-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular structure of an apolipoprotein determined at 2.5-A resolution

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.     

Crystal structure of a biologically inactive mutant of toxic shock syndrome toxin-1 at 2.5 A resolution.

Toxic shock syndrome toxin-1 (TSST-1) is one of a family of staphylococcal exotoxins recognized as microbial superantigens. The toxin plays a dominant role in the genesis of toxic shock in humans through a massive activation of the immune system. This potentially lethal illness occurs as a result of the interaction of TSST-1 with a significant proportion of the T-cell repertoire. TSST-1, like other superantigens, can bind directly to class II major histocompatibility (MHC class II) molecules prior to its interaction with entire families of V beta chains of the T-cell receptor (TCR). The three-dimensional structure of a mutant (His-135-Ala) TSST-1 was compared with the structure of the native (wild-type) TSST-1 at 2.5 A resolution. The replacement of His 135 of TSST-1 with an Ala residue results in the loss of T-cell mitogenicity and toxicity in experimental animals. This residue, postulated to be directly involved in the toxin-TCR interactions, is located on the major helix alpha 2, which forms the backbone of the molecule and is exposed to the solvent. In the molecular structure of the mutant toxin, the helix alpha 2 remains unaltered, but the His to Ala modification causes perturbations on the neighboring helix alpha 1 by disrupting helix-helix interactions. Thus, the effects on TCR binding of the His 135 residue could actually be mediated, wholly or in part, by the alpha 1 helix.     

Crystal structure of human recombinant interleukin-4 at 2.25 A resolution.

The crystal structure of human recombinant interleukin-4 (IL-4) has been solved by multiple isomorphous replacement, and refined to an R factor of 0.218 at 2.25 A resolution. The molecule is a left-handed four-helix bundle with a short stretch of beta sheet. The structure bears close resemblance to other cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF). Although no sequence similarity of IL-4 to GM-CSF and other related cytokines has been previously postulated, structure-based alignment of IL-4 and GM-CSF revealed that the core of the molecules, including large parts of all four helices and extending over half of the molecule, has 30% sequence identity. This may have identified regions which are not only important to maintain structure, but could also play a role in receptor binding.     

Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution

The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com.     

Beta-lactamase TEM1 of E. coli. Crystal structure determination at 2.5 A resolution.

The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.     

Crystal structure of thermitase at 1.4 A resolution

The crystal structure of thermitase, a subtilisin-type serine proteinase from Thermoactinomyces vulgaris, was determined by X-ray diffraction at 1.4 A resolution. The structure was solved by a combination of molecular and isomorphous replacement. The starting model was that of subtilisin BPN' from the Protein Data Bank, determined at 2.5 A resolution. The high-resolution refinement was based on data collected using synchrotron radiation with a Fuji image plate as detector. The model of thermitase refined to a conventional R factor of 14.9% and contains 1997 protein atoms, 182 water molecules and two Ca ions. The tertiary structure of thermitase is similar to that of the other subtilisins although there are some significant differences in detail. Comparison with subtilisin BPN' revealed two major structural differences. The N-terminal region in thermitase, which is absent in subtilisin BPN', forms a number of contacts with the tight Ca2+ binding site and indeed provides the very tight binding of the Ca ion. In thermitase the loop of residues 60 to 65 forms an additional (10) beta-strand of the central beta-sheet and the second Ca2+ binding site that has no equivalent in the subtilisin BPN' structure. The observed differences in the Ca2+ binding and the increased number of ionic and aromatic interactions in thermitase are likely sources of the enhanced stability of thermitase.     

Crystal structure of lactoperoxidase at 2.4 A resolution

Lactoperoxidase (LPO) is a member of the mammalian peroxidase superfamily. It catalyzes the oxidation of thiocyanate and halides. Freshly isolated and purified samples of caprine LPO were saturated with ammonium iodide and crystallized using 20% polyethylene glycol 3350 in a hanging drop vapor diffusion setup. The structure has been determined using X-ray crystallographic method and refined to R_cryst and R_free factors of 0.196 and 0.203, respectively. The structure determination revealed an unexpected phosphorylation of Ser198 in LPO, which is also confirmed by anti-phosphoserine antibody binding studies. The structure is also notable for observing densities for glycan chains at all the four potential glycosylation sites. Caprine LPO consists of a single polypeptide chain of 595 amino acid residues and folds into an oval-shaped structure. The structure contains 20 well-defined α-helices of varying lengths including a helix, H_2a, unique to LPO, and two short antiparallel β-strands. The structure confirms that the heme group is covalently linked to the protein through two ester linkages involving carboxylic groups of Glu258 and Asp108 and modified methyl groups of pyrrole rings A and C, respectively. The heme moiety is slightly distorted from planarity, but pyrrole ring B is distorted considerably. However, an iron atom is displaced only by 0.1 Å from the plane of the heme group toward the proximal site. The substrate diffusing channel in LPO is cylindrical in shape with a diameter of approximately 6 Å. Two histidine residues and six buried water molecules are connected through a hydrogen-bonded chain from the distal heme cavity to the surface of protein molecule and seemingly form the basis of proton relay for catalytic action. Ten iodide ions have been observed in the structure. Out of these, only one iodide ion is located in the distal heme cavity and is hydrogen bonded to the water molecule W1. W1 is also hydrogen bonded to the heme iron as well as to distal His109. The structure contains a calcium ion that is coordinated to seven oxygen atoms and forms a typical pentagonal bipyramidal coordination geometry.     

The structure of yeast enolase at 2.25-A resolution. An 8-fold beta + alpha-barrel with a novel beta beta alpha alpha (beta alpha)6 topology

The three-dimensional structure of yeast enolase has been determined by the multiple isomorphous replacement method followed by the solvent flattening technique. A polypeptide model, corresponding with the known amino acid sequence, has been fitted to the electron density map. Crystallographic restrained least-squares refinement of the model without solvent gave R = 20.0% for 6-2.25-A resolution with good geometry. A model with 182 water molecules and 1 sulfate which is still being refined has presently R = 17.0%. The molecule is a dimer with subunits related by 2-fold crystallographic symmetry. The subunit has dimensions 60 X 55 X 45 A and is built from two domains. The smaller N-terminal domain has an alpha + beta structure based on a three-stranded antiparallel meander and four helices. The main domain is an 8-fold beta + alpha-barrel. The enolase barrel is, however, different from the triose phosphate isomerase barrel; its topology is beta beta alpha alpha (beta alpha)6 rather than (beta alpha)8 as found in triose phosphate isomerase. The inner beta-barrel is not entirely parallel, the second strand is antiparallel to the other strands, and the direction of the first helix is also reversed with respect to the other helices. This supports the hypothesis that some enzymes evolved independently producing the stable structure of beta alpha barrels with either enolase or triose phosphate isomerase topology. The active site of enolase is located at the carboxylic end of the barrel. A fragment of the N-terminal domain and two long loops protruding from the barrel domain form a wide crevice leading to the active site region. Asp246, Glu295, and Asp320 are the ligands of the conformational cation. Other residues in the active site region are Glu168, Asp321, Lys345, and Lys396.     

Crystal structure of the trigonal form of bovine beta-lactoglobulin and of its complex with retinol at 2.5 A resolution

The structure of the trigonal crystal form of bovine beta-lactoglobulin has been determined by X-ray diffraction methods. An electron density map, calculated with phases obtained by the multiple isomorphous replacement method, served as a starting point for alternate cycles of model building and restrained least-squares refinement. The model of the molecule fitted to the initial Fourier map was the one built for the orthorhombic crystal form of beta-lactoglobulin, solved at 2.8 A resolution (1 A = 0.1 nm). The final R factor for 1456 atoms (1276 non-hydrogen protein atoms and 180 solvent atoms) is 0.22, including 5245 reflections from 6.0 to 2.5 A. The molecule shows significant differences in the two crystal forms mentioned, mainly due to different packing. In the trigonal form, the species crystallized does not appear to be dimeric, but a linear polymer with tight intermolecular contacts. A difference electron density map between the complex of beta-lactoglobulin with retinol and the native protein shows no significant peaks in the cavity which, in the similar retinol-binding protein, binds the chromophore. Instead, differences are found at a surface pocket, which is limited almost completely by hydrophobic residues.