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 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 study on human S100A13 at 2.0 A resolution

The S100 protein family is the largest group of calcium-binding protein families, which consists of at least 25 members. S100A13, which is widely expressed in a variety of tissues, is a unique member of the S100 protein family. Previous reports showed that S100A13 might be involved in the stress-induced release of some signal peptide-less proteins (such as FGF-1 and IL-1alpha) and also associated with inflammatory functions. It was also reported that S100A13 is a new angiogenesis marker. Here we report the crystal structure of the Ca(2+)-bound form of S100A13 at 2.0 A resolution. S100A13 is a homodimer with four EF-hand motifs in an asymmetric unit, displaying a folding pattern similar to other S100 members. However, S100A13 has the unique structural feature with all alpha-helices being amphiphilic, which was not found in other members of S100s. We propose that this characteristic structure of S100A13 might be related to its ability to mediate the release of FGF-1 and IL-1alpha.     

Crystal structure of a secretory signalling glycoprotein from sheep at 2.0A resolution

A 40kDa glycoprotein from dry secretion of sheep is implicated as a signaling factor and is named as SPS-40. This protein is secreted only during the early phase of involution when the drastic tissue remodeling occurs in the mammary gland. SPS-40 was purified from sheep dry secretions and crystallized using hanging drop vapour diffusion method. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell dimensions, a=62.7A, b=66.4A, c=107.5A. The protein was also cloned for the determination of its complete amino acid sequence. The three-dimensional structure of SPS-40 was determined by X-ray crystallographic method at 2.0A resolution. The structure revealed the presence of an N-linked glycan chain at Asn39. The protein adopts a conformation with a classical (beta/alpha)(8)-barrel fold of triosephosphate isomerase (TIM) (residues 1-237 and 310-360) with an insertion of a small (alpha+beta) domain (residues 240-307) similar to that observed in chitinases. However, the Leu substitution for Glu in the consensus catalytic sequence in SPS-40 causes a loss of chitinase activity. Furthermore, the sugar-binding groove in SPS-40 is distorted considerably from the standard chitin-binding site in chitinase enzymes and hence the binding of chitin-like oligosaccharides is considerably hampered. Three surface loops, His188-His197, Phe202-Arg212 and Phe244-Pro260 have exceptionally high values of B-factors (average=70.5A(2)), indicating the presence of a less defined region.     

Crystal structure of N-acyl-D-glucosamine 2-epimerase from porcine kidney at 2.0 A resolution

The X-ray crystallographic structure of N-acyl-d-glucosamine 2-epimerase (AGE) from porcine kidney, which has been identified to be a renin-binding protein (RnBP), was determined by the multiple isomorphous replacement method and refined at 2.0 A resolution with a final R-factor of 16.9 % for 15 to 2.0 A resolution data. The refined structure of AGE comprised 804 amino acid residues (one dimer) and 145 water molecules. The dimer of AGE had an asymmetric unit with approximate dimensions 46 Ax48 Ax96 A. The AGE monomer is composed of an alpha(6)/alpha(6)-barrel, the structure of which is found in glucoamylase and cellulase. One side of the AGE alpha(6)/alpha(6)-barrel structure comprises long loops containing five short beta-sheets, and contributes to the formation of a deep cleft shaped like a funnel. The putative active-site pocket and a possible binding site for the substrate N-acetyl-d-glucosamine (GlcNAc) were found in the cleft. The other side of the alpha(6)/alpha(6)-barrel comprises short loops and contributes to the dimer formation. At the dimer interface, which is composed of the short loops and alpha-helices of the subunits, five strong ion-pair interactions were observed, which play a major role in the dimer assembly. This completely ruled out the previously accepted hypothesis that the formation of the RnBP homodimer and RnBP-renin heterodimer requires the leucine zipper motif present in RnBP.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 A resolution

Cytidine deaminases (CDA, EC 3.5.4.5) are zinc-containing enzymes in the pyrimidine salvage pathway that catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. Two different classes have been identified in the CDA family, a homodimeric form (D-CDA) with two zinc ions per dimer and a homotetrameric form (T-CDA) with four zinc ions per tetramer. We have determined the first structure of a T-CDA from Bacillus subtilis. The active form of T-CDA is assembled of four identical subunits with one active site apiece. The subunit of D-CDA is composed of two domains each exhibiting the same fold as the T-CDA subunits, but only one of them contains zinc in the active site. The similarity results in a conserved structural core in the two CDA forms. An intriguing difference between the two CDA structures is the zinc coordinating residues found at the N-terminal of two alpha-helices: three cysteine residues in the tetrameric form and two cysteine residues and one histidine residue in the dimeric form. The role of the zinc ion is to activate a water molecule and thereby generate a hydroxide ion. How the zinc ion in T-CDA surrounded with three negatively charged residues can create a similar activity of T-CDA compared to D-CDA has been an enigma. However, the structure of T-CDA reveals that the negative charge caused by the three ligands is partly neutralized by (1) an arginine residue hydrogen-bonded to two of the cysteine residues and (2) the dipoles of two alpha-helices.     

Crystal structure of alginate lyase A1-III complexed with trisaccharide product at 2.0 A resolution

The structure of A1-III from a Sphingomonas species A1 complexed with a trisaccharide product (4-deoxy-l-erythro-hex-4-enepyranosyluronate-mannuronate-mannuronic acid) was determined by X-ray crystallography at 2.0 A with an R-factor of 0.16. The final model of the complex form comprising 351 amino acid residues, 245 water molecules, one sulfate ion and one trisaccharide product exhibited a C(alpha) r.m.s.d. value of 0.154 A with the reported apo form of the enzyme. The trisaccharide was bound in the active cleft at subsites -3 approximately -1 from the non-reducing end by forming several hydrogen bonds and van der Waals interactions with protein atoms. The catalytic residue was estimated to be Tyr246, which existed between subsites -1 and +1 based on a mannuronic acid model oriented at subsite +1.     

Crystal structure of an Escherichia coli tRNA(Gly) microhelix at 2.0 A resolution

tRNA identity elements determine the correct aminoacylation by the cognate aminoacyl-tRNA synthetase. In class II aminoacyl tRNA synthetase systems, tRNA specificity is assured by rather few and simple recognition elements, mostly located in the acceptor stem of the tRNA. Here we present the crystal structure of an Escherichia coli tRNA(Gly) aminoacyl stem microhelix at 2.0 A resolution. The tRNA(Gly) microhelix crystallizes in the space group P3(2)21 with the cell constants a=b=35.35 A, c=130.82 A, gamma=120 degrees . The helical parameters, solvent molecules and a potential magnesium binding site are discussed.     

Crystal and molecular structure of the bovine alpha-chymotrypsin-eglin c complex at 2.0 A resolution.

The crystal structure of the complex between bovine alpha-chymotrypsin and the leech (Hirudo medicinalis) protein proteinase inhibitor eglin c has been refined at 2.0 A resolution to a crystallographic R-factor of 0.167. The structure of the complex includes 2290 protein and 143 solvent atoms. Eglin c is bound to the cognate enzyme through interactions involving 11 residues of the inhibitor (sites P5-P4' in the reactive site loop, P10' and P23') and 17 residues from chymotrypsin. Binding of eglin c to the enzyme causes a contained hinge-bending movement around residues P4 and P4' of the inhibitor. The tertiary structure of chymotrypsin is little affected, with the exception of the 10-13 region, where an ordered structure for the polypeptide chain is observed. The overall binding mode is consistent with those found in other serine proteinase-protein-inhibitor complexes, including those from different inhibition families. Contained, but significant differences are observed in the establishment of intramolecular hydrogen bonds and polar interactions stabilizing the structure of the intact inhibitor, if the structure of eglin c in its complex with chymotrypsin is compared with that of other eglin c-serine proteinase complexes.     

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.     

Crystal structure of a transcarbamylase-like protein from the anaerobic bacterium Bacteroides fragilis at 2.0 A resolution

A transcarbamylase-like protein essential for arginine biosynthesis in the anaerobic bacterium Bacteroides fragilis has been purified and crystallized in space group P4(3)2(1)2 (a=b=153.4 A, c=94.8 A). The structure was solved using a single isomorphous replacement with anomalous scattering (SIRAS) and was refined at 2.0 A resolution to an R-factor of 20.6% (R-free=25.2%). The molecular model is trimeric and comprises 960 amino acid residues, two phosphate groups and 422 water molecules. The monomer has the consensus transcarbamylase fold with two structural domains linked by two long interdomain helices: the putative carbamoyl phosphate-binding domain and a binding domain for the second substrate. Each domain has a central parallel beta-sheet surrounded by alpha-helices and loops with alpha/beta topology. The putative carbamoyl phosphate-binding site is similar to those in ornithine transcarbamylases (OTCases) and aspartate transcarbamylases (ATCases); however, the second substrate-binding site is strikingly different. This site has several insertions and deletions, and residues critical to substrate binding and catalysis in other known transcarbamylases are not conserved. The three-dimensional structure and the fact that this protein is essential for arginine biosynthesis suggest strongly that it is a new member of the transcarbamylase family. A similar protein has been found in Xylella fastidiosa, a bacterium that infects grapes, citrus and other plants.     

Crystal structure of the stromelysin catalytic domain at 2.0 A resolution: inhibitor-induced conformational changes.

Matrix metalloproteinases are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. Stromelysin is a zinc-dependent proteinase and a member of the matrix metalloproteinase family. We have solved the crystal structure of an active uninhibited form of truncated stromelysin and a complex with a hydroxamate-based inhibitor. The catalytic domain of the enzyme of residues 83-255 is an active fragment. Two crystallographically independent molecules, A and B, associate as a dimer in the crystals. There are three alpha-helices and one twisted, five-strand beta-sheet in each molecule, as well as one catalytic Zn, one structural Zn and three structural Ca ions. The active site of stromelysin is located in a large, hydrophobic cleft. In particular, the S1' specificity site is a deep and highly hydrophobic cavity. The structure of a hydroxamate-phosphinamide-type inhibitor-bound stromelysin complex, formed by diffusion soaking, has been solved as part of our structure-based design strategy. The most important feature we observed is an inhibitor-induced conformational change in the S1' cavity which is triggered by Tyr223. In the uninhibited enzyme structure, Tyr223 completely covers the S1' cavity, while in the complex, the P1' group of the inhibitor displaces the Tyr223 in order to fit into the S1' cavity. Furthermore, the displacement of Tyr223 induces a major conformational change of the entire loop from residue 222 to residue 231. This finding provides direct evidence that Tyr223 plays the role of gatekeeper of the S1' cavity. Another important intermolecular interaction occurs at the active sit of molecule A, in which the C-terminal tail (residues 251-255) from molecule B inserts. The C-terminal tail interacts extensively with the active site of molecule A, and the last residue (Thr255) coordinated to the catalytic zinc as the fourth ligand, much like a product inhibitor would. The inhibitor-induced conformational change and the intermolecular C-terminal-zinc coordination are significant in understanding the structure-activity relationships of the enzyme.     

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.     

Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution.

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 A resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 A resolution with residual 15.5%. The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure. The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.     

The refined crystal structure of ribonuclease A at 2.0 A resolution

This paper describes the structure of bovine pancreatic ribonuclease A, refined by a restrained parameter least squares procedure at 2.0 A resolution, and rebuilt using computer graphics. The final agreement factor (formula see text) is 0.159. The positions of the 951 main chain atoms have been determined with an estimated accuracy of 0.17 A. In addition, the model includes a phosphate group in the active site and 176 waters, many of them with partial occupancy. The bond lengths in the refined structure of RNase A differ from the ideal values by an overall root mean square deviation of 0.022 A; the corresponding value for angle distances is 0.06 A. The root mean square deviation of planar atoms from ideality is 0.017 A, and root mean square deviation of the peptide torsion angles from 180 degrees is 3.4 degrees. The model is in good agreement with the final difference Fourier maps. Two active site histidines, His 12 and His 119, form hydrogen bonds to the phosphate ion. His 119 is also hydrogen bonded to the carboxyl of ASp 121 and His 12 to the carbonyl of Thr 45. The structure of the RNase A is very similar to that of RNase S, particularly in the active site region. The root mean square discrepancy of all atoms from residues 1 to 16 and 24 to 123 is 1.06 A and the root mean square discrepancy for the active site region is 0.6 A.     

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.