Refined structure of bovine carbonic anhydrase III at 2.0 Å resolution

The three-dimensional structure of bovine carbonic anhydrase III (BCA III) from red skeletal muscle cells has been determined by molecular replacement methods. The structure has been refined at 2.0 Å resolution by both constrained and restrained structure-factor least squares refinement. The current crystallographic R-value is 19.2% and 121 solvent molecules have so far been found associated with the protein. The structure is highly similar to the refined structure of human carbonic anhydrase II. Some differences in amino acid sequence and structure between the two isoenzymes are discussed. In BCA III, Lys 64 and Arg 91 (His 64 and Ile 91 in HCA II) are both pointing out from the active site cavity forming salt bridges with Glu 4 and Asp 72 (His 4 and Asp 72 in HCA II), respectively. However, Arg 67 and Phe 198 (Asn 67 and Leu 198 in HCA II) are oriented towards the zinc ion and significantly reduce the volume of the active site cavity. Phe 198 particularly reduces the size of the substrate binding region at the “deep water” position at the bottom of the cavity and we sugest that this is one of the major reasons for the differences in catalytic properties of isoenzyme III as compared to isozyme II. © 1993 Wiley-Liss, Inc.     

Crystal structure of R22L trichosanthin

Ｔｒｉｃｈｏｓａｎｔｈｉｎ(ＴＣＳ)ｉｓａｎｉｍｐｏｒｔａｎｔｍｅｍｂｅｒｏｆｒｉｂｏｓｏｍｅｉｎａｃｔｉｖａｔｉｎｇｐｒｏｔｅｉｎｓ[1].ＩｔｐｏｓｓｅｓｓｅｓＮｇｌｙｃｏｓｉｄａｓｅａｃｔｉｖｉｔｙｒｅｍｏｖｉｎｇａｄｅｎｉｎｅ(ＡＤＥ)ａｔｐｏｓｉｔｉｏｎＡ4324ｏｆ28ＳｒＲＮＡ[2].ＴｈｅａｃｔｉｖｅｐｏｃｋｅｔｏｆＮｇｌｙｃｏｓｉｄａｓｅｈａｓｂｅｅｎｅｓｔａｂｌｉｓｈｅｄｔｈｒｏｕｇｈｔｈｅｃｒｙｓｔａｌｓｔｒｕｃｔｕｒｅｓｏｆＴＣＳ,αＭＭＣａｎｄｒｉｃｉｎａｎｄａｓｓａｙｏｆｍｕｔａｎｔｓ…     

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis

Summary Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing multiple pharmacological properties. One of its interesting properties is to inhibit human immunodeficiency virus (HIV) replication but its strong immunogenicity has limited the repeated clinical administration. To map the antigenic determinants and reduce the immunogenicity of TCS, two potential antigenic sites (YFF81–83 and KR173–174) were identified by computer modeling, and then three TCS mutants namely TCS_{YFF81–83ACS}, TCS_{KR173–174CG}, and TCS_YFF-KR were constructed by site-directed mutagenesis. The RI activity and DNase-like activity of the three constructed TCS mutants were similar to natural TCS but with much lower immunogenicity. Results suggested that the two selected sites are all located at or near the antigenic determinants of TCS. In toxicity studies, the LD₅₀ of the three TCS mutants was not different from natural TCS. These findings would be useful in designing a better therapeutic agent for AIDS.Qunxing An and Sanhua Wei equally contribute to this work.     

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.     

Refined structure of human carbonic anhydrase II at 2.0 Å resolution

The structure of human erythrocytic carbonic anhydrase II has been refined by constrained and restrained structure–factor least-squares refinement at 2.0 Å resolution. The conventional crystallographic R value is 17.3%. Of 167 solvent molecules associated with the protein, four are buried and stabilize secondary structure elements. The zinc ion is ligated to three histidyl residues and one water molecule in a nearly tetrahedral geometry. In addition to the zinc-bound water, seven more water molecules are identified in the active site. Assuming that Glu-106 is deprotonated at pH 8.5, some of the hydrogen bond donor–acceptor relations in the active site can be assigned and are described here in detail. The Oγ1 atom of Thr-199 donates its proton to the Oε1 atom of Glu-106 and can function as a hydrogen bond acceptor only in additional hydrogen bonds.     

Crystal structure of TGF-β2 refined at 1.8 Å resolution

The crystal structure of TGF-β2 has been refined using data collected with synchrotron radiation (CHESS) to 1.8 Å resolution with a residual R (= ∑ | |F_o| ? |F_c| | /∑ |F_o|) factor of 17.3%. The model consists of 890 protein atoms from all 112 residues and 59 water molecules. The monomer of TGF-β2 assumes a rather extended conformation and lacks a well-defined hydrophobic core. Surface accessibility calculations show only 44% of the nonpolar surface is buried in the monomer. In contrast, 55.8% of the nonpolar surface area is buried when the two monomers from a dimer, a typical value for globular proteins. This includes a 1300 Ų buried interface area that is largely hydrophobic. Sequence comparisons using a profile derived from the refined TGF-β2 structure suggest that the cluster of four disulfides (three intramonomeric disulfide bonds 15–78, 44–109, 48–111 forming a disulfide knot, and one intermonomeric disulfide 77–77) together with the extended β strand region constitutes the conserved structural motif for the TGF-β superfamily. This structural motif, without the 77–77 disulfide bond, defines also the common fold for a general family of growth factors, including the nerve growth factor and platelet-derived growth factor families. The fold is conserved only at the monomer level, while the active forms are dimers, suggesting that dimerization plays an important role in regulating the binding of these cytokines to their receptors and in modulating the biological responses. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Crystal structure of the invertebrate bifunctional purine biosynthesis enzyme PAICS at 2.8 Å resolution

Two important steps of the de novo purine biosynthesis pathway are catalyzed by the 5‐aminoimidazole ribonucleotide carboxylase and the 4‐(N‐succinylcarboxamide)‐5‐aminoimidazole ribonucleotide synthetase enzymes. In most eukaryotic organisms, these two activities are present in the bifunctional enzyme complex known as PAICS. We have determined the 2.8‐Å resolution crystal structure of the 350‐kDa invertebrate PAICS from insect cells (Trichoplusia ni) using single‐wavelength anomalous dispersion methods. Comparison of insect PAICS to human and prokaryotic homologs provides insights into substrate binding and reveals a highly conserved enzymatic framework across divergent species. Proteins 2013; 81:1473–1478. © 2013 Wiley Periodicals, Inc.     

The structure of porin from Rhodobacter capsulatus at 1.8 Å resolution

The structure of the porin from Rhodobacter capsulanus was determined at a resolution of 1.8 Å. The analysis started from a closely related crystal structure that had been solved at a medium resolution of 3 Å using multiple isomorphous replacement and solvent flattening. The new structure contains the complete sequence of 301 amino acid residues. Refinement of the model is under way: the present R-factor is 22% with good geometry. Except for the lengths of several loops, the resulting chain fold corresponds to the medium resolution model. The membrane channel is lined by a large number of ionogenic side chains with characteristic segregation of differently charged groups.     

Prediction of antigenic determinants of trichosanthin by molecular modeling

The antigenic determinants of trichosanthin were predicted by molecular modeling.First,the threedimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence.Secondly,the preferable antigen-antibody interactions were obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E.Two regions in the molecular surface of trichosanthin were found to form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antigenic determinants:one is composed of two polypeptide segments,Ile201-Glu210 and Ile225-Asp229,which are close to each other in the three-dimensional structure;and the other is the segment Lys173-Thr178.The former region seems to be the more reasonable antigenic determinant than the latter one.     

Structure of the extremely slow GTPase Rab6A in the GTP bound form at 1.8 Å resolution

Rab/Ypt GTPases represent a>60 member large family of membrane traffic regulators in eukaryotic cells. Members of this group display intrinsic GTPase activity varying over two orders of magnitude. Here, we show that Rab6A represents the RabGTPase with the slowest spontaneous GTPase activity yet measured (5x10(-6)s(-1)). Due to the very low intrinsic hydrolysis rate we were able to crystallise and solve the structure of the Rab6A:GTP complex to 1.82A resolution. Analysis of the structure suggests that low catalytic activity of the Rab6A might be due to high flexibility of the Switch II region and a low degree of constraint of critically important for catalysis Gln 72.     

Structure of a ternary complex of proteinase K,mercury, and a substrate-analogue hexa-peptide at 2.2 Å resolution

The crystal structure of a ternary complex of proteinase K, Hg(II) and a hexapeptide N-Ac-Pro-Ala-Pro-Phe-Pro-Ala-NH₂ has been determined at 2.2 Å resolution and refined to an R factor of 0.172 for 12,910 reflections. The mercury atom occupies two alternate sites, each of which was assigned an occupancy of 0.45. These two sites are bridged by Cys-73 Sγ which forms covalent bonds to both. Both mercury sites form regular polyhedrons involving atoms from residues Asp-39, His-69, Cys-73, His-72, Met-225, and Wat-324. The complex formation with mercury seems to disturb the stereochemistry of the residues of the catalytic triad Asp-39, His-69, and Ser-224 appreciably, thus reducing the enzymatic activity of proteinase K to 15%. The electron density in the difference Fourier map shows that the hexapeptide occupies the S1 subsite predominantly and the standard recognition site constituted by Ser-132 to Gly-136 and Gly-100 to Tyr-104 segments is virtually empty. The hexapeptide is held firmly through a series of hydrogen bonds involving protein atoms and water molecules. As a result of complex formation, Asp-39, His-69, Met-225, Ile-220, Ser-219, Thr-223, and Ser-224 residues move appreciably to accommodate the mercury atoms and the hexapeptide. The largest movement is observed for Met-225 which is involved in multiple interactions with both mercury and the hexapeptide. The activity results indicate an inhibition rate of 95%, as a result of the combined effect of mercury and hexapeptide. © 1996 Wiley-Liss, Inc.     

Crystal structure of Escherichia coli RNase HI in complex with Mg2+ at 2.8 Å resolution: Proof for a single Mg2+-binding site

To obtain more precise insight into the Mg²⁺-binding site essential for RNase HI catalytic activity, we have determined the crystal structure of E. coli RNase HI in complex with Mg²⁺. The analyzed cocrystal, which is not isomorphous with the Mg²⁺-free crystal previously refined at 1.48 Å resolution, was grown at a high MgSO₄ concentration more than 100 mM so that even weakly bound Mg²⁺ sites could be identified. The structure was solved by the molecular replacement method, using the Mg²⁺-free crystal structure as a search model, and was refined to give a final R-value of 0.190 for intensity data from 10 to 2.8 Å, using the XPLOR and PROLSQ programs. The backbone structures are in their entirety very similar to each other between the Mg²⁺-bound and the metal-free crystals, except for minor regions in the enzyme interface with the DNA/RNA hybrid. The active center clearly revealed a single Mg²⁺ atom located at a position almost identical to that previously found by the soaking method. Although the two metal-ion mechanism had been suggested by another group (Yang, W., Hendrickson, W.A., Crouch, R.J., Satow, Y. Science 249:1398-1405, 1990) and partially supported by the crystallographic study of inactive HIV-1 RT RNase H fragment (Davies, J.F., II, Hostomska, Z., Hostomsky, Z., Jordan, S.R., Matthews, D. Science 252:88-95, 1991), the present result excludes the possibility that RNase HI requires two metal-binding sites for activity. In contrast to the features in the metal-free enzyme, the side chains of Asn-44 and Glu-48 are found to form coordinate bonds with Mg²⁺ in the metal-bound crystal. © 1993 Wiley-Liss, Inc.     

Crystallographic structure of metal-free concanavalin A at 2.5 Å resolution

The three-dimensional structure of demetallized concanavalin A has been determined at 2.5 Å resolution and refined to a crystallographic R-factor of 18%. The lectin activity of concanavalin A requires the binding of both a transition metal ion, generally Mn²⁺, and a Ca²⁺ ion in two neighboring sites in close proximity to the carbohydrate binding site. Large structural differences between the native and the metal-free lectin are observed in the metal-binding region and consequently for the residues involved in the specific binding of saccharides. The demetallization invokes a series of conformational changes in the protein backbone, apparently initiated mainly by the loss of the calcium ion. Most of the Mn²⁺ ligands retain their position, but the Ca²⁺ binding site is destroyed. The Ala207-Asp208 peptide bond, in the β-strand neighboring the metal-binding sites, undergoes a cis to trans isomerization. The cis conformation for this bond is a highly conserved feature among the leguminous lectins and is critically maintained by the Ca²⁺ ion in metal-bound concanavalin A. A further and major change adjacent to the isomerized bond is an expansion of the loop containing the monosaccharide ligand residues Leu99 and Tyr100. The dispersion of the ligand residues for the monosaccharide binding site (Asn14, Agr228, Asp208, Leu99, and Tyr100) in metalfree concanavalin A abolishes the lectin's ability to bind saccharides. Since the quaternary structure of legume lectins is essential to their biological role, the tetramer formation was analyzed. In the crystal (pH 5), the metal-free concanavalin A dimers associate into a tetramer that is similar to the native one, but with a drastically reduced number of inter-dimer interactions. This explains the tetramer dissociation into dimers below pH values of 6.5. © 1995 Wiley-Liss, Inc.     

Change in pH-dependent membrane insertion characteristics of trichosanthin caused by deletion of its last seven C-terminal amino acid residues

Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) that can selectively kill some types of cells at low concentration (0.1-1 nM). The pH-dependent membrane insertion ability of TCS makes it possible that the internalized toxin avoids degradation in lysosomes and further undergoes transportation into the cytosol by some still unidentified mechanism. Here, we show that deletion of C-terminal residues affects interactions of modified TCS (C7-TCS) with lipids and reduces its pH-dependent membrane insertion ability. Fluorescence measurements indicate that at low pH C7-TCS undergoes profound conformational changes that causes exposure of a hydrophobic region and leads to oligomerization of the C7-TCS molecules. The results suggest that the membrane insertion of TCS at low pH might be important for translocation of TCS into the cytosol, which is important for exertion of the RIP activity of TCS. Deletion of the last seven C-terminal residues of TCS would reduce both its RIP activity in vitro and cytotoxicity in vivo, with the degree of decrease being more significant for the cytotoxicity in vivo.     

Structure of Paramecium tetraurelia calmodulin at 1.8 A resolution.

The crystal structure of calmodulin (CaM; M(r) 16,700, 148 residues) from the ciliated protozoan Paramecium tetraurelia (PCaM) has been determined and refined using 1.8 A resolution area detector data. The crystals are triclinic, space group P1, a = 29.66, b = 53.79, c = 25.49 A, alpha = 92.84, beta = 97.02, and gamma = 88.54 degrees with one molecule in the unit cell. Crystals of the mammalian CaM (MCaM; Babu et al., 1988) and Drosophila CaM (DCaM; Taylor et al., 1991) also belong to the same space group with very similar cell dimensions. All three CaMs have 148 residues, but there are 17 sequence changes between PCaM and MCaM and 16 changes between PCaM and DCaM. The initial difference in the molecular orientation between the PCaM and MCaM crystals was approximately 7 degrees as determined by the rotation function. The reoriented Paramecium model was extensively refitted using omit maps and refined using XPLOR. The R-value for 11,458 reflections with F > 3 sigma is 0.21, and the model consists of protein atoms for residues 4-147, 4 calcium ions, and 71 solvent molecules. The root mean square (rms) deviations in the bond lengths and bond angles in the model from ideal values are 0.016 A and 3 degrees, respectively. The molecular orientation of the final PCaM model differs from MCaM by only 1.7 degrees. The overall Paramecium CaM structure is very similar to the other calmodulin structures with a seven-turn long central helix connecting the two terminal domains, each containing two Ca-binding EF-hand motifs. The rms deviation in the backbone N, Ca, C, and O atoms between PCaM and MCaM is 0.52 A and between PCaM and DCaM is 0.85 A. The long central helix regions differ, where the B-factors are also high, particularly in PCaM and MCaM. Unlike the MCaM structure, with one kink at D80 in the middle of the linker region, and the DCaM structure, with two kinks at K75 and I85, in our PCaM structure there are no kinks in the helix; the distortion appears to be more gradually distributed over the entire helical region, which is bent with an apparent radius of curvature of 74.5(2) A. The different distortions in the central helical region probably arise from its inherent mobility.     

Design of anti‐thyroid drugs: Binding studies and structure determination of the complex of lactoperoxidase with 2‐mercaptoimidazole at 2.30 Å resolution

Lactoperoxidase (LPO) belongs to mammalian heme peroxidase superfamily, which also includes myeloperoxidase (MPO), eosinophil peroxidase (EPO), and thyroid peroxidase (TPO). LPO catalyzes the oxidation of a number of substrates including thiocyanate while TPO catalyzes the biosynthesis of thyroid hormones. LPO is also been shown to catalyze the biosynthesis of thyroid hormones indicating similar functional and structural properties. The binding studies showed that 2‐mercaptoimidazole (MZY) bound to LPO with a dissociation constant of 0.63 µM. The inhibition studies showed that the value of IC₅₀ was 17 µM. The crystal structure of the complex of LPO with MZY showed that MZY bound to LPO in the substrate‐binding site on the distal heme side. MZY was oriented in the substrate‐binding site in such a way that the sulfur atom is at a distance of 2.58 Å from the heme iron. Previously, a similar compound, 3‐amino‐1,2,4‐triazole (amitrole) was also shown to bind to LPO in the substrate‐binding site on the distal heme side. The amino nitrogen atom of amitrole occupied the same position as that of sulfur atom in the present structure indicating a similar mode of binding. Recently, the structure of the complex of LPO with a potent antithyroid drug, 1‐methylimidazole‐2‐thiol (methimazole, MMZ) was also determined. It showed that MMZ bound to LPO in the substrate‐binding site on the distal heme side with 2 orientations. The position of methyl group was same in the 2 orientations while the positions of sulfur atom differed indicating a higher preference for a methyl group.