Crystal structure of spinach plastocyanin at 1.7 A resolution.

The crystal structure of plastocyanin from spinach has been determined using molecular replacement, with the structure of plastocyanin from poplar as a search model. Successful crystallization was facilitated by site-directed mutagenesis in which residue Gly8 was substituted with Asp. The region around residue 8 was believed to be too mobile for the wild-type protein to form crystals despite extensive screening. The current structure represents the oxidized plastocyanin, copper (II), at low pH (approximately 4.4). In contrast to the similarity in the core region as compared to its poplar counterpart, the structure shows some significant differences in loop regions. The most notable is the large shift of the 59-61 loop where the largest shift is 3.0 A for the C(alpha) atom of Glu59. This results in different patterns of electrostatic potential around the acidic patches for the two proteins.     

Crystal structure of human annexin I at 2.5 A resolution.

cDNA coding for N-terminally truncated human annexin I, a member of the family of Ca(2+)-dependent phospholipid binding proteins, has been cloned and expressed in Escherichia coli. The expressed protein is biologically active, and has been purified and crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 139.36 A, b = 67.50 A, and c = 42.11 A. The crystal structure has been determined by molecular replacement at 3.0 A resolution using the annexin V core structure as the search model. The average backbone deviation between these two structures is 2.34 A. The structure has been refined to an R-factor of 17.7% at 2.5 A resolution. Six calcium sites have been identified in the annexin I structure. Each is located in the loop region of the helix-loop-helix motif. Two of the six calcium sites in annexin I are not occupied in the annexin V structure. The superpositions of the corresponding loop regions in the four domains show that the calcium binding loops in annexin I can be divided into two classes: type II and type III. Both classes are different from the well-known EF-hand motif (type I).     

The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells.

Thioltransferase (glutaredoxin) was purified from human red blood cells essentially as described previously (Mieyal JJ et al., 1991a, Biochemistry 30:6088-6097). The primary sequence of the HPLC-pure enzyme was determined by tandem mass spectrometry and found to represent a 105-amino acid protein of molecular weight 11,688 Da. The physicochemical and catalytic properties of this enzyme are common to the group of proteins called glutaredoxins among the family of thiol:disulfide oxidoreductases that also includes thioredoxin and protein disulfide isomerase. Although this human red blood cell glutaredoxin (hRBC Grx) is highly homologous to the 3 other mammalian Grx proteins whose sequences are known (calf thymus, rabbit bone marrow, and pig liver), there are a number of significant differences. Most notably an additional cysteine residue (Cys-7) occurs near the N-terminus of the human enzyme in place of a serine residue in the other proteins. In addition, residue 51 of hRBC Grx displayed a mixture of Asp and Asn. This result is consistent with isoelectric focusing analysis, which revealed 2 distinct bands for either the oxidized or reduced forms of the protein. Because the enzyme was prepared from blood combined from a number of individual donors, it is not clear whether this Asp/Asn ambiguity represents inter-individual variation, gene duplication, or a deamidation artifact of purification.     

Crystal structure of thermitase from Thermoactinomyces vulgaris at 2.2 A resolution

The crystal structure of thermitase from Thermoactinomyces vulgaris has been determined by x-ray diffraction at 2.2 A resolution. The structure was solved by a combination of single isomorphous replacement and molecular replacement methods. The structure was refined to a conventional R factor of 0.24 using restrained least square procedures CORELS and PROLSQ. The tertiary structure of thermitase is similar to that of subtilsin BPN'. The greatest differences between these structures are related to the insertions and deletions in the sequence.     

Crystal structure of human coactosin-like protein at 1.9 A resolution

Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 A resolution. The structure features a central beta-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed.     

Crystal structure of adenosine kinase from Toxoplasma gondii at 1.8 A resolution

Human infection with Toxoplasma gondii is an important cause of morbidity and mortality. Protozoan parasites such as T. gondii are incapable of de novo purine biosynthesis and must acquire purines from their host, so the purine salvage pathway offers a number of potential targets for antiparasitic chemotherapy. In T. gondii tachyzoites, adenosine is the predominantly salvaged purine nucleoside, and thus adenosine kinase is a key enzyme in the purine salvage pathway of this parasite. The structure of T. gondii adenosine kinase was solved using molecular replacement and refined by simulated annealing at 1.8 A resolution to an R-factor of 0.214. The overall structure and the active site geometry are similar to human adenosine kinase, although there are significant differences. The T. gondii adenosine kinase has several unique features compared to the human sequence, including a five-residue deletion in one of the four linking segments between the two domains, which is probably responsible for a major change in the orientation of the two domains with respect to each other. These structural differences suggest the possibility of developing specific inhibitors of the parasitic enzyme.     

Crystal structure of beta-amylase from Bacillus cereus var. mycoides at 2.2 A resolution.

The crystal structure of beta-amylase from Bacillus cereus var. mycoides was determined by the multiple isomorphous replacement method. The structure was refined to a final R-factor of 0.186 for 102,807 independent reflections with F/sigma(F) > or = 2.0 at 2.2 A resolution with root-mean-square deviations from ideality in bond lengths, and bond angles of 0.014 A and 3.00 degrees, respectively. The asymmetric unit comprises four molecules exhibiting a dimer-of-dimers structure. The enzyme, however, acts as a monomer in solution. The beta-amylase molecule folds into three domains; the first one is the N-terminal catalytic domain with a (beta/alpha)8 barrel, the second one is the excursion part from the first one, and the third one is the C-terminal domain with two almost anti-parallel beta-sheets. The active site cleft, including two putative catalytic residues (Glu172 and Glu367), is located on the carboxyl side of the central beta-sheet in the (beta/alpha)8 barrel, as in most amylases. The active site structure of the enzyme resembles that of soybean beta-amylase with slight differences. One calcium ion is bound per molecule far from the active site. The C-terminal domain has a fold similar to the raw starch binding domains of cyclodextrin glycosyltransferase and glucoamylase.     

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.     

Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2-A resolution.

The crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) at 2.2-A resolution has been determined by the molecular replacement method. The crystal belongs to space group R32 with cell parameters a = b = 105.3 A, c = 189.4 A, alpha = beta = 90 degrees, gamma = 120 degrees. After several cycles of refinement using program X-PLOR and model building based on the electron density map, the crystallographic R-factor converged to 19.3% (R-free factor is 26.9%) in the range of 10.0 to 2.2 A. The r.m.s. deviations of bond length and angles are 0.015 A and 2.9 degrees, respectively. In the crystal, two APC-PY trimers associate face to face into a hexamer. The assembly of two trimers within the hexamer is similar to that of C-phycocyanin (C-PC) and R-phycoerythrin (R-PE) hexamers, but the assembly tightness of the two trimers to the hexamer is not so high as that in C-PC and R-PE hexamers. The chromophore-protein interactions and possible pathway of energy transfer were discussed. Phycocyanobilin 1alpha84 of APC-PY forms 5 hydrogen bonds with 3 residues in subunit 2beta of another monomer. In R-PE and C-PC, chromophore 1alpha84 only forms 1 hydrogen bond with 2beta77 residue in subunit 2beta. This result may support and explain great spectrum difference exists between APC trimer and monomer.     

Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.     

Crystal structure of a proteolytically generated functional monoferric C-lobe of bovine lactoferrin at 1.9A resolution

This is the first crystal structure of a proteolytically generated functional C-lobe of lactoferrin. The purified samples of iron-saturated C-lobe were crystallized in 0.1 M Mes buffer (pH 6.5) containing 25% (v/v) polyethyleneglycol monomethyl ether 550 M and 0.1 M zinc sulphate heptahydrate. The X-ray intensity data were collected with 300 mm imaging plate scanner mounted on a rotating anode generator. The structure was determined by the molecular replacement method using the coordinates of the C-terminal half of bovine lactoferrin as a search model and refined to an R-factor of 0.193 for all data to 1.9A resolution. The final model comprises 2593 protein atoms (residues 342-676 and 681-685), 124 carbohydrate atoms (from ten monosaccharide units, in three glycan chains), one Fe(3+), one CO(3)(2-), two Zn(2+) and 230 water molecules. The overall folding of the C-lobe is essentially the same as that of C-terminal half of bovine lactoferrin but differs slightly in conformations of some of the loops and reveals a number of new interactions. There are 20 Cys residues in the C-lobe forming ten disulphide links. Out of these, one involving Cys481-Cys675 provides an inter-domain link at 2.01A while another Cys405-Cys684 is formed between the main C-lobe 342-676 and the hydrolyzed pentapeptide 681-685 fragment. Six inter-domain hydrogen bonds have been observed in the structure whereas only four were reported in the structure of intact lactoferrin, although domain orientations have been found similar in the two structures. The good quality of electron density has also revealed all the ten oligosaccharide units in the structure. The observation of two metal ions at sites other than the iron-binding cleft is another novel feature of the present structure. These zinc ions stabilize the crystal packing. This structure is also notable for extensive inter-molecular hydrogen bonding in the crystals. Therefore, the present structure appears to be one of the best packed crystal structures among the proteins of the transferrin superfamily.     

Crystal structure of recombinant human tissue kallikrein at 2.0 A resolution.

Human tissue kallikrein, a trypsin-like serine protease involved in blood pressure regulation and inflammation processes, was expressed in a deglycosylated form at high levels in Pichia pastoris, purified, and crystallized. The crystal structure at 2.0 A resolution is described and compared with that of porcine kallikrein and of other trypsin-like proteases. The active and S1 sites (nomenclature of Schechter I, Berger A, 1967, Biochem Biophys Res Commun 27:157-162) are similar to those of porcine kallikrein. Compared to trypsin, the S1 site is enlarged owing to the insertion of an additional residue, cis-Pro 219. The replacement Tyr 228 --> Ala further enlarges the S1 pocket. However, the replacement of Gly 226 in trypsin with Ser in human tissue kallikrein restricts accessibility of substrates and inhibitors to Asp 189 at the base of the S1 pocket; there is a hydrogen bond between O delta1Asp189 and O gammaSer226. These changes in the architecture of the S1 site perturb the binding of inhibitors or substrates from the modes determined or inferred for trypsin. The crystal structure gives insight into the structural differences responsible for changes in specificity in human tissue kallikrein compared with other trypsin-like proteases, and into the structural basis for the unusual specificity of human tissue kallikrein in cleaving both an Arg-Ser and a Met-Lys peptide bond in its natural protein substrate, kininogen. A Zn+2-dependent, small-molecule competitive inhibitor of kallikrein (Ki = 3.3 microM) has been identified and the bound structure modeled to guide drug design.     

Crystal structure of the extracellular domain of mouse RANK ligand at 2.2-A resolution.

Bone remodeling involves the resorption of bone by osteoclasts and the synthesis of bone matrix by osteoblasts. Receptor activator of NF-kappa B ligand (RANKL, also known as ODF and OPGL), a member of the tumor necrosis factor (TNF) family, triggers osteoclastogenesis by forming a complex with its receptor, RANK. We have determined the crystal structure of the extracellular domain of mouse RANKL at 2.2-A resolution. The structure reveals that the RANKL extracellular domain is trimeric, which was also shown by analytical ultracentrifugation, and each subunit has a beta-strand jellyroll topology like the other members of the TNF family. A comparison of RANKL with TNF beta and TNF-related apoptosis-inducing ligand (TRAIL), whose structures were determined to be in the complex form with their respective receptor, reveals conserved and specific features of RANKL in the TNF superfamily and suggests the presence of key residues of RANKL for receptor binding.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Crystal structure of (L-Arg)-BO bovine insulin at 0.21 nm resolution

The crystal structure of (L-Arg)-B0 bovine insulin has been determined, using data to 0.21 nm and atomic parameters of 2Zn porcine insulin as a starting model, by the difference Fourier method, the restrained least square method and X-PLOR package, interspersed with careful review of the electron density, to a final R-factor of 0.182 and r.m.s. deviation of 0.002 2nm for the bond lengths and 4.3° for the bond angles. The electron densities of additional (L-Arg)-B0 residues to B-chain N-terminus of two monomers in each asymmetric unit are very dear. The crystallographic micro-environment of the N-terminus of the B-chain is different from that of rhombohedral 2-zinc insulin.     

Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes.

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.     

Crystal structure of echicetin from Echis carinatus (Indian saw-scaled viper) at 2.4A resolution

Echicetin is a heterodimeric protein from the venom of the Indian saw-scaled viper, Echis carinatus. It binds to platelet glycoprotein Ib (GPIb) and thus inhibits platelet aggregation. It has two subunits, alpha and beta, consisting of 131 and 123 amino acid residues, respectively. The two chains are linked with a disulphide bond. The level of amino acid sequence homology between two subunits is 50%. The protein was purified from the venom of E.carinatus and crystallized using ammonium sulphate as a precipitant. The crystal structure has been determined at 2.4A resolution and refined to an R-factor of 0.187. Overall dimensions of the heterodimer are approximately 80Ax35Ax35A. The backbone folds of the two subunits are similar. The central portions of the polypeptide chains of alpha and beta-subunits move into each other to form a tight dimeric association. The remaining portions of the chains of both subunits fold in a manner similar to those observed in the carbohydrate-binding domains of C-type lectins. In echicetin, the Ca(2+)-binding sites are not present, despite being topologically equivalent to other similar Ca(2+)-binding proteins of the superfamily. The residues Ser41, Glu43 and Glu47 in the calcium-binding proteins of the related family are conserved but the residues Glu126/120 are replaced by lysine at the corresponding sites in the alpha and beta-subunits.     

Three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.