Structure of interleukin 1 alpha at 2.7-A resolution

The interleukin 1 (IL-1) family of proteins has a central role in modulating immune and inflammatory responses. Two major IL-1 proteins, designated alpha (IL-1 alpha) and beta (IL-1 beta), are produced by activated macrophages and other cell types. In an effort to understand the similarities and differences in the physicochemical and functional properties of these two proteins, a program was initiated to determine their structures. Crystals of IL-1 alpha were grown, and the three-dimensional structure at 2.7-A resolution was solved. The technique of multiple-wavelength anomalous dispersion (MAD) with the selenomethionine form of IL-1 alpha was utilized in combination with a single mercury derivative to provide the starting phases. Partial refinement of the IL-1 alpha model has been performed as well. The overall structure is composed of 14 beta-strands and a 3(10) helix. The core of this structure is a capped beta-barrell that possesses 3-fold symmetry and displays a topology similar to that observed for IL-1 beta [Priestle, J. P., et al. (1988) EMBO J. 7, 339-343] and soybean trypsin inhibitor (STI) [McLachlan, A. D. (1979) J. Mol. Biol. 133, 557-563]. In this paper, the overall structure of IL-1 alpha and the nature and fidelity of the internal 3-fold symmetry are discussed. Comparisons with IL-1 beta and STI are made within these contexts.     

Structure of acid protease from Endothia parasitica in cross-linked form at 2.45-A resolution.

The structure of acid protease from Endothia parasitica in strongly cross-linked form is compared with that of the untreated protein at 2.45-a resolution. The only observed conformation change introduced by the cross-linking reaction is at the N terminal. Otherwise the two main chain structures are essentially identical. Approximately 2 molecules of the inhibitor, 1,2-epoxy-3-(p-nitrophenoxy)propane, are found to be incorporated into each protein molecule. They are covalently bound to the two aspartic residues at the active center.     

Structure of the concanavalin A-methyl alpha-D-mannopyranoside complex at 6-A resolution.

The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A-methyl alpha-D-mannopyranoside complex and is 35 A from the iodophenol binding site (K. D. Hardman and C. F. Ainsworth (1973), Biochemistry 12,4442), which has been postulated to be adjacent to the carbohydrate-specific binding site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 2580). The crystals are orthorhombic in space group C222(1) and crystal denisty measurements indicate a protein mass of four monomers (molecular weight of 104 000) per asymmetric unit. However, the electron density map contains eight monomers/asymmetric unit, revealing lattice disorder. The electron density map with a nominal resolution of 6 A has been solved using three heavy-atom derivatives and the position and orientation of each monomer established. Atomic coordinates of the native protein which has previously been determined (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103) were transposed into this new space group and the gross conformations of the monomers, dimers, and tetramers were found to be very similar to the previous structure. However, some minor differences were apparent even at this resolution. After crystal growth, the methyl alpha-D-mannopyranoside was replaced by o-iodophenyl beta-D-glucopyranoside or methyl 2-iodoacetimido-2-deoxy-alpha-D-glucopyranoside in separate experiments, and difference electron density maps were calculated. The highest peaks for both iodinated sugar derivatives associated with each monomer agreed within a few angstroms of each other and were found near side chains Tyr-12 and -100 and Asp-16 and -208. This region is 10-14 A from the manganese, in good agreement with nuclear magnetic resonance (NMR) studies in solution (C. F. Brewer et al. (1973), Biochemistry 12, 4448) and with the site predicted from crosslinked 1222 crystal studies (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103).     

Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.     

Structure of yeast triosephosphate isomerase at 1.9-A resolution

The structure of yeast triosephosphate isomerase (TIM) has been solved at 3.0-A resolution and refined at 1.9-A resolution to an R factor of 21.0%. The final model consists of all non-hydrogen atoms in the polypeptide chain and 119 water molecules, a number of which are found in the interior of the protein. The structure of the active site clearly indicates that the carboxylate of the catalytic base, Glu 165, is involved in a hydrogen-bonding interaction with the hydroxyl of Ser 96. In addition, the interactions of the other active site residues, Lys 12 and His 95, are also discussed. For the first time in any TIM structure, the "flexible loop" has well-defined density; the conformation of the loop in this structure is stabilized by a crystal contact. Analysis of the subunit interface of this dimeric enzyme hints at the source of the specificity of one subunit for another and allows us to estimate an association constant of 10(14)-10(16) M-1 for the two monomers. The analysis also suggests that the interface may be a particularly good target for drug design. The conserved positions (20%) among sequences from 13 sources ranging on the evolutionary scale from Escherichia coli to humans reveal the intense pressure to maintain the active site structure.     

The molecular structure of the left-handed Z-DNA double helix at 1.0-A atomic resolution. Geometry, conformation, and ionic interactions of d(CGCGCG)

The structure of d(CGCGCG) crystallized in the presence of magnesium and sodium ions alone is compared to that of the spermine form of the molecule. The very high resolution nature of these structure determinations allows the first true examination of an oligonucleotide structure in fine detail. The values of bond distances and angles are compared to those derived from small molecule crystal structures. In addition, the interactions of cations and polyamines with the Z-DNA helix are analyzed. In particular, multiple cationic charges appear to offer enhanced stabilization for the Z-DNA conformation. The location of spermine molecules along the edge of the deep groove and also spanning the entrance to the groove emphasizes the importance of polyamines for stabilizing this left-handed structure. On averaging, we obtained very similar structural parameters for the two different structures with standard deviations generally smaller than the deviations of the crystallographic model from ideal values. This indicates a high degree of accuracy of the two structures, which have been refined using different data and different refinement methods. The derived bond lengths and angles may thus be more representative of this polymeric DNA structure than those derived from mono- and dinucleotide structures at a similar accuracy.     

The structure of human lymphotoxin (tumor necrosis factor-beta) at 1.9-A resolution.

The three-dimensional structure of recombinant human lymphotoxin (residues 24-171 of the mature protein) has been determined by x-ray crystallography at 1.9-A resolution (Rcryst = 0.215 for I greater than 3 sigma (I)). Phases were derived by molecular replacement using tumor necrosis factor (TNF-alpha) as a search model. Like TNF-alpha, lymphotoxin (LT) folds to form a "jellyroll" beta-sheet sandwich. Three-fold related LT subunits form a trimer stabilized primarily by hydrophobic interactions. A cluster of 6 basic residues around the 3-fold axis may account for the acid lability of the trimer. Although the structural cores of TNF-alpha and LT are similar, insertions and deletions relative to TNF-alpha occur in loops at the "top" of the LT trimer and significantly alter the local structure and the overall shape trimer is highly conserved. The sites of two mutations (Asp-50 and Tyr-108) that abolish the cytotoxicity of LT are contained within poorly ordered loops of polypeptide chain that flank the cleft between neighboring subunits at the base of the molecule, suggesting that the receptor recognizes an intersubunit binding site.     

Crystal structure of a Z-DNA hexamer d(CGCICG) at 1.7 A resolution: inosine.cytidine base-pairing, and comparison with other Z-DNA structures.

The crystal structure of the deoxyhexamer, d(CGCICG), has been determined and refined to a resolution of 1.7A. The DNA hexamer crystallises in space group P2(1)2(1)2(1) with unit cell dimensions of a = 18.412 +/- .017 A, b = 30.485 +/- .036A, and c = 43.318 +/- .024 A. The structure has been solved by rotation and translation searches and refined to an R-factor of 0.148 using 2678 unique reflections greater than 1.0 sigma (F) between 10.0-1.7 A resolution. Although the crystal parameters are similar to several previously reported Z-DNA hexamers, this inosine containing Z-DNA differs in the relative orientation, position, and crystal packing interactions compared to d(CGCGCG) DNA. Many of these differences in the inosine form of Z-DNA can be explained by crystal packing interactions, which are responsible for distortions of the duplex at different locations. The most noteworthy features of the inosine form of Z-DNA as a result of such distortions are: (1) sugar puckers for the inosines are of C4'-exo type, (2) all phosphates have the Zl conformation, and (3) narrower minor grove and compression along the helical axis compared to d(CGCGCG) DNA. In addition, the substitution of guanosine by inosine appears to have resulted in Watson-Crick type base-pairing between inosine and cytidine with a potential bifurcated hydrogen bond between inosine N1 and cytidine N3 (2.9 A) and O2 (3.3-3.A).     

The structure of prothrombin fragment 1 at 3.5-A resolution

The structure of prothrombin fragment 1 has been determined at 3.5-A resolution by multiple isomorphous replacement methods with four heavy atom derivatives. The final average figure of merit is 0.72. There is a large cylindrical solvent region with an average diameter of 35-40 A along the entire length of the c axis (85 A) centered at about x = y = 1/2. The connected density forming the wall of this channel is not of sufficient extent to account for the 156 residues of fragment 1 and the two accompanying carbohydrate chains totaling 5000 in molecular weight. Deglycosylated fragment 1 crystallizes isomorphously with fragment 1, and a difference map between the two revealed that the sugar chains are severely disordered and reside in the solvent channel. Although the disordered carbohydrate and the complexity of five disulfides in a 126-residue sequence have hampered the complete tracing of the peptide chain, two-thirds of the molecule has been accounted for in the form of an unusually oblate ellipsoid of about 15 X 30 X 35 A. The folding of the molecule has little secondary structure (one alpha-helix (7%), 20% beta-structure) in agreement with dichroism measurements and one of the points of carbohydrate attachment is suggested from the deglycosylated difference map.     

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.     

Three-dimensional structure of p-cresol methylhydroxylase (flavocytochrome c) from Pseudomonas putida at 3.0-A resolution.

p-Cresol methylhydroxylase (PCMH) isolated from Pseudomonas putida is an alpha 2 beta 2 tetramer of approximate subunit Mr 49,000 and 9,000. It is a flavocytochrome c containing covalently bound FAD in the larger subunit and covalently bound heme in the smaller. Crystals in space group P2(1)2(1)2(1) with unit-cell parameters a = 140.3 A, b = 130.6 A, and c = 74.1 A contain one full molecule per asymmetric unit and diffract anisotropically to about 2.8-A resolution in two directions and to about 3.3-A resolution in the third. An electron density map has been computed at a nominal resolution of 3.0 A by use of area detector data from native crystals and from two derivatives. The phases were improved with the B.C. Wang solvent leveling procedure, and the map was averaged about the noncrystallographic 2-fold axis. The cytochrome subunit, whose amino acid sequence is known, has been fitted to the electron density on a graphics system. The course of the polypeptide chain of the flavoprotein subunit, whose sequence is mostly unknown, has been traced in a minimap and a model of polyalanine fitted to the electron density on the graphics system. The flavoprotein subunit consists of three domains in close contact. The N-terminal domain consists largely of beta-structure and contains most of the FAD binding site. The second domain contains a seven-stranded antiparallel beta-sheet of unusual topology connected by antiparallel alpha-helices on one side. The flavin ring lies at the juncture of the first two domains. The third domain lies against the first domain and helps cover the rest of the FAD chain. The cytochrome subunit resembles other small cytochromes such as c-551 and c5 and fits into a depression on the surface of the large flavoprotein subunit. The flavin and heme planes are nearly perpendicular, the normals to the planes being approximately 65 degrees apart. The two groups are separated by about 8 A, the distance from one of the vinyl methylene carbon atoms of the heme to the 8 alpha-methyl group of the flavin ring.     

Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution

The crystal structure of calmodulin (Mr 16,700, 148 residues) from Drosophila melanogaster as expressed in a bacterial system has been determined and refined at 2.2-A resolution. Starting with the structure of mammalian calmodulin, we produced an extensively refitted and refined model with a conventional crystallographic R value of 0.197 for the 5,239 reflections (F greater than or equal to 2 sigma (F)) within the 10.0-2.2-A resolution range. The model includes 1,164 protein atoms, 4 calcium ions, and 78 water molecules and has root mean square deviations from standard values of 0.018 A for bond lengths and 0.043 A for angle distances. The overall structure is similar to mammalian calmodulin, with a seven-turn central helix connecting the two calcium-binding domains. The "dumb-bell" shaped molecule contains seven alpha-helices and four "EF hand" calcium-binding sites. Although the amino acid sequences of mammalian and Drosophila calmodulins differ by only three conservative amino acid changes, the refined model reveals a number of significant differences between the two structures. Superimposition of the structures yields a root mean square deviation of 1.22 A for the 1,120 equivalent atoms. The calcium-binding domains have a root mean square deviation of 0.85 A for the 353 equivalent atoms. There are also differences in the amino terminus, the bend of the central alpha-helix, and the orientations of some of the side chains.     

Structure of Plasmodium falciparum triose-phosphate isomerase-2-phosphoglycerate complex at 1.1-A resolution

Triose-phosphate isomerase, a key enzyme of the glycolytic pathway, catalyzes the isomerization of dihydroxy acetone phosphate and glyceraldehyde 3-phosphate. In this communication we report the crystal structure of Plasmodium falciparum triose-phosphate isomerase complexed to the inhibitor 2-phosphoglycerate at 1.1-A resolution. The crystallographic asymmetric unit contains a dimeric molecule. The inhibitor bound to one of the subunits in which the flexible catalytic loop 6 is in the open conformation has been cleaved into two fragments presumably due to radiation damage. The cleavage products have been tentatively identified as 2-oxoglycerate and meta-phosphate. The intact 2-phosphoglycerate bound to the active site of the other subunit has been observed in two different orientations. The active site loop in this subunit is in both open and "closed" conformations, although the open form is predominant. Concomitant with the loop closure, Phe-96, Leu-167, and residues 208-211 (YGGS) are also observed in dual conformations in the B-subunit. Detailed comparison of the active-site geometry in the present case to the Saccharomyces cerevisiae triose-phosphate isomerase-dihydroxy acetone phosphate and Leishmania mexicana triose-phosphate isomerase-phosphoglycolate complexes, which have also been determined at atomic resolution, shows that certain interactions are common to the three structures, although 2-phosphoglycerate is neither a substrate nor a transition state analogue.     

Conformational perturbation of the anticancer nucleotide arabinosylcytosine on Z-DNA: molecular structure of (araC-dG)3 at 1.3 A resolution.

The left-handed Z-DNA structure of an araC-containing (where araC stands for arabinosylcytosine) hexamer, (araC-dG)3, has been solved by x-ray diffraction analysis at 1.3 A resolution. This hexamer was crystallized in the hexagonal P6(5)22 (a = b = 17.96 A, c = 43.22 A) space group in which the hexamers have statistically disordered packing arrangement along the 6(5) screw axis, yet the crystals diffract x-rays to high resolution. Its structure has been refined by the constrained least square refinement to a final R factor of 0.287 using 737 [> 3.0 sigma(F)] observed reflections. The asymmetric unit of the unit cell contains only a dinucleotide, 5'-p (araC)p(dG). The overall conformation resembles that of the canonical Z-DNA, but with some differences in details. The O2' hydroxyl groups of the araC residues form intramolecular hydrogen bonds with N2 of the 5'-guanine residues. In the deep groove of Z-DNA, these hydroxy groups replace the bridging water molecules that stabilize the guanine in the syn conformation. The results reinforce the earlier observation made by the structural analysis of another hexamer, d(CG[araC]GCG), with a mono-substitution of araC [M.-K. Teng, Y.-C. Liaw, G. A. van der Marel, J. H. van Boom, and A. H.-J. Wang (1989) Biochemistry, vol. 28, pp. 4923-4928]. These two structures show that araC residue can be incorporated readily into the Z structure and probably facilitates the B to Z transition, as supported by uv absorption spectroscopic studies in a number of araC-containing oligonucleotides. The potential biological roles of the araC-modified Z-DNA are discussed.     

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

Bromination stabilizes poly(dG-dC) in the Z-DNA form under low-salt conditions

Using circular dichroism studies, Pohl & Jovin (1972) [Pohl, F.M., & Jovin, T.M. (1972) J. Mol. Biol. 67, 375-396] demonstrated that poly(dG-dC) undergoes a salt-dependent conformational change characterized by a spectral inversion. The low-salt form corresponds to the right-handed B form of DNA and the high-salt form to the left-handed Z-DNA helix. Modification of poly(dG-dC) by adding bromine atoms to the C8 position of guanine and the C5 position of cytosine residues stabilized this polymer in the Z-DNA form under low-salt conditions. The guanine residues were found to be twice as reactive as the cytosine residues. With a modification of 38% Br8G and 18% Br5C, the polymers formed a stable Z-DNA helix under physiological conditions. The bromination produced spectroscopic features very similar to poly(dG-dC) in 4 M NaCl. However, bromination did not freeze the Z structure as was shown by ethidium bromide intercalation studies. Addition of the dye favored an intercalated B-DNA form. The conversion of B- to Z-DNA leads to profound conformational changes which were also seen by a reduced insensitivity to various exo- and endonucleases. Comparative studies showed that the brominated polymers have a high affinity to nitrocellulose filters. In 1 M NaCl, there was virtually no binding of B-DNA, but a substantial binding of Z-DNA was found even at rather low levels of bromination.(ABSTRACT TRUNCATED AT 250 WORDS)