Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution

The enzyme ribonuclease T1 (RNase T1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2',5'-guanosine (2',5'-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3 sigma in the resolution range 6 to 1.8 A. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T1/2',5'-GpG complex and of the near-isomorphous RNase T1/2'-GMP complex are comparable. The root-mean-square deviation is 0.73 A between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5'-terminal guanine of 2',5'-GpG binds to RNase T1 in much the same way as in the 2'-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3'-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2',5'-GpG (except the 5'-terminal guanine), which originates from differences in the pucker of the 5'-terminal ribose. The pucker is C2'-exo for the major site (2/3 occupancy) and C1'-endo for the minor site (1/3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3'-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2',5'-GpG, which exceeds that of all other inhibitors of type 2',5'-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.     

Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-A resolution

The atomic resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations. Also for the side chain of the catalytic glutamate, Glu(167), two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of the ligand and the catalytic glutamate. The distance between O11 of PGA and Oepsilon2 of Glu(167) is 2.61 and 2.55 A for the major and minor conformations, respectively. In either conformation, Oepsilon1 of Glu(167) is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually exclusive arrangements. Despite the structural disorder in the active site, the C termini of the beta strands that construct the active site display the least anisotropy compared with the rest of the protein. The loops following these beta strands display various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro(168) at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.     

Three-dimensional structure of the ribonuclease T1 2'-GMP complex at 1.9-A resolution

The complex formed between the enzyme ribonuclease T1 (EC 3.1.27.3) and its specific inhibitor 2'-guanylic acid (2'-GMP) has been refined to R = 0.180 using x-ray diffraction data to 1.9-A resolution. The protein molecule displays a compact fold; a 4.5 turn alpha-helix packed over an antiparallel beta-pleated sheet shields most of the hydrophobic interior of the protein against the solvent. The extended pleated sheet structure of ribonuclease T1 is composed of three long and four short strands building up a two-stranded minor beta-sheet near the amino terminus and a five-stranded major sheet in the interior of the protein molecule. In the complex with ribonuclease T1, the inhibitor 2'-guanylic acid adopts the syn-conformation and C2'-endo sugar pucker. Binding of the nucleotide is mainly achieved through amino acid residues 38-46 of the protein. The catalytically active amino acid residues of ribonuclease T1 (His40, Glu58, Arg77, and His92) are located within the major beta-sheet which, as evident from the analysis of atomic temperature factors, provides an environment of minimal local mobility. The geometry of the active site is consistent with a mechanism for phosphodiester hydrolysis where, in the transesterification step, His40 and/or Glu58 act as a general base toward the ribose 2'-hydroxyl group and His92, as a general acid, donates a proton to the leaving 5'-hydroxyl group.     

The refined crystal structure of a fully active semisynthetic ribonuclease at 1.8-A resolution

A fully active, semisynthetic analog of bovine ribonuclease A, comprised of residues 1-118 of the molecule in a noncovalent complex with the synthetic peptide analog of residues 111-124, has been crystallized in space group P3(2)21 from a solution of 1.3 M ammonium sulfate and 3.0 M cesium chloride at pH 5.2. The crystallographic structure was determined by rotation and translation searches utilizing the coordinates for ribonuclease A reported by Wlodawer and Sjolin (Wlodawer, A., and Sjolin, L. (1983) Biochemistry 22, 2720-2728) and has been refined at 1.8-A resolution to an agreement factor of 0.204. Most of the structure of the semisynthetic enzyme closely resembles that found in ribonuclease A with the synthetic peptide replacing the C-terminal elements of the naturally occurring enzyme. No redundant structure is seen; residues 114-118 of the larger chain and residues 111-113 of the peptide do not appear in our map. The positions of those residues at or near the active site are very similar to, if not identical with, those previously reported by others, except for histidine 119, which occupies predominantly the B position seen as a minor site by Borkakoti et al. (Borkakoti, N., Moss, D. S., and Palmer, R. A. (1982) Acta Crystallogr. Sect. B Struct. Crystallogr. Cryst. Chem. 38,2210-2217) and not at all by Wlodawer and Sjolin (1983).     

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 at 1.5-A resolution of Pyrus pyrifolia pistil ribonuclease responsible for gametophytic self-incompatibility

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S(3)-RNase) responsible for gametophytic self-incompatibility was determined at 1.5-A resolution. It consists of eight helices and seven beta-strands, and its folding topology is typical of RNase T(2) family enzymes. Based on a structural comparison of S(3)-RNase with RNase Rh, a fungal RNase T(2) family enzyme, the active site residues of S(3)-RNase assigned were His(33) and His(88) as catalysts and Glu(84) and Lys(87) as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short alpha-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

Crystal structure of a phycourobilin-containing phycoerythrin at 1.90-A resolution.

The structure of R-phycoerythrin (R-PE) from the red alga Griffithsia monilis was solved at 1.90-A resolution by molecular replacement, using the atomic coordinates of cyanobacterial phycocyanin from Fremyella diplosiphon as a model. The crystallographic R factor for the final model is 17.5% (Rfree 22.7%) for reflections in the range 100-1.90 A. The model consists of an (alphabeta)2 dimer with an internal noncrystallographic dyad and a fragment of the gamma-polypeptide. The alpha-polypeptide (164 amino acid residues) has two covalently bound phycoerythrobilins at positions alpha82 and alpha139. The beta-polypeptide (177 residues) has two phycoerythrobilins bound to residues beta82 and beta158 and one phycourobilin covalently attached to rings A and D at residues beta50 and beta61, respectively. The electron density of the gamma-polypeptide is mostly averaged out by threefold crystallographic symmetry, but a dipeptide (Gly-Tyr) and one single Tyr could be modeled. These two tyrosine residues of the gamma-polypeptide are in close proximity to the phycoerythrobilins at position beta82 of two symmetry-related beta-polypeptides and are related by the same noncrystallographic dyad as the (alphabeta)2 dimer. Possible energy transfer pathways are discussed briefly.     

The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-A resolution

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 A by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.     

Crystal structure of recombinant rabbit interferon-gamma at 2.7-A resolution

The crystal structure of recombinant rabbit interferon-gamma was solved by the multiple isomorphous replacement technique at 2.7-A resolution and refined to a crystallographic R-factor of 26.2%. The interferon crystallizes with one-half of the functional dimer in the asymmetric unit, with the two polypeptide chains of the dimer related by a crystallographic 2-fold symmetry axis. The structure is predominantly alpha-helical with extensive interdigitation of the alpha-helical segments of the two polypeptide chains.     

Crystal structure of human parathyroid hormone 1-34 at 0.9-A resolution

The N-terminal fragment 1-34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1-34) and determined the structure to 0.9-A resolution. hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1-34) and hPTHrP-(1-34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1-34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1-34) are proposed.     

Crystal structure of AlgQ2, a macromolecule (alginate)-binding protein of Sphingomonas sp. A1, complexed with an alginate tetrasaccharide at 1.6-A resolution

Sphingomonas sp. A1 possesses a high molecular weight (HMW) alginate uptake system composed of a novel pit formed on the cell surface and a pit-dependent ATP-binding cassette (ABC) transporter in the inner membrane. The transportation of HMW alginate from the pit to the ABC transporter is mediated by the periplasmic HMW alginate-binding proteins AlgQ1 and AlgQ2. We determined the crystal structure of AlgQ2 complexed with an alginate tetrasaccharide using an alginate-free (apo) form as a search model and refined it at 1.6-A resolution. One tetrasaccharide was found between the N and C-terminal domains, which are connected by three extended hinge loops. The tetrasaccharide complex took on a closed domain form, in contrast to the open domain form of the apo form. The tetrasaccharide was bound in the cleft between the domains through van der Waals interactions and the formation of hydrogen bonds. Among the four sugar residues, the nonreducing end residue was located at the bottom of the cleft and exhibited the largest number of interactions with the surrounding amino acid residues, suggesting that AlgQ2 mainly recognizes and binds to the nonreducing part of a HMW alginate and delivers the polymer to the ABC transporter through conformational changes (open and closed forms) of the two domains.     

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 macrophage migration inhibitory factor complexed with (E)-2-fluoro-p-hydroxycinnamate at 1.8 A resolution: implications for enzymatic catalysis and inhibition.

Macrophage migration inhibitory factor (MIF) exhibits dual activities. It acts as an immunoregulatory protein as well as a phenylpyruvate tautomerase. To understand better the relationship between these two activities and to elucidate the structural basis for the enzymatic activity, a crystal structure of a complex between murine MIF and (E)-2-fluoro-p-hydroxycinnamate, a competitive inhibitor of the tautomerase activity, has been determined to 1.8 A resolution. The structure is nearly superimposable on that of the free protein indicating that the presence of the inhibitor does not result in any major structural changes. The inhibitor also confirms the location of the active site in a hydrophobic cavity containing the amino-terminal proline. Within this cavity, the inhibitor interacts with residues from adjacent subunits. At the back of the cavity, the side-chain carbonyl oxygen of Asn-97' interacts with the phenolic hydroxyl group of the inhibitor while at the mouth of the cavity the ammonium group of Lys-32 interacts with a carboxylate oxygen. The other carboxylate oxygen of the inhibitor interacts with Pro-1. The hydroxyl group of Tyr-95' interacts weakly with the fluoro group on the inhibitor. The hydrophobic side chains of five active-site residues (Met-2, Ile-64, Met-101, Val-106, and Phe-113) and the phenyl moiety of Tyr-95' are responsible for the binding of the phenyl group. Further insight into the enzymatic activity of MIF was obtained by carrying out kinetic studies using the enol isomers of phenylpyruvate and (p-hydroxyphenyl)pyruvate. The results demonstrate that MIF processes the enol isomers more efficiently than the keto isomers primarily because of a decrease in Km. On the basis of these results, a mechanism is proposed for the MIF-catalyzed tautomerization reaction.     

X-ray structure of Escherichia coli pyridoxine 5'-phosphate oxidase complexed with FMN at 1.8 A resolution

BACKGROUND: Escherichia coli pyridoxine 5'-phosphate oxidase (PNPOx) catalyzes the terminal step in the biosynthesis of pyridoxal 5'-phosphate (PLP), a cofactor used by many enzymes involved in amino acid metabolism. The enzyme oxidizes either the 4'-hydroxyl group of pyridoxine 5'-phosphate (PNP) or the 4'-primary amine of pyridoxamine 5'-phosphate (PMP) to an aldehyde. PNPOx is a homodimeric enzyme with one flavin mononucleotide (FMN) molecule non-covalently bound to each subunit. A high degree of sequence homology among the 15 known members of the PNPOx family suggests that all members of this group have similar three-dimensional folds. RESULTS: The crystal structure of PNPOx from E. coli has been determined to 1.8 A resolution. The monomeric subunit folds into an eight-stranded beta sheet surrounded by five alpha-helical structures. Two monomers related by a twofold axis interact extensively along one-half of each monomer to form the dimer. There are two clefts at the dimer interface that are symmetry-related and extend from the top to the bottom of the dimer. An FMN cofactor that makes interactions with both subunits is located in each of these two clefts. CONCLUSIONS: The structure is quite similar to the recently deposited 2.7 A structure of Saccharomyces cerevisiae PNPOx and also, remarkably, shares a common structural fold with the FMN-binding protein from Desulfovibrio vulgaris and a domain of chymotrypsin. This high-resolution E. coli PNPOx structure permits predictions to be made about residues involved in substrate binding and catalysis. These predictions provide testable hypotheses, which can be answered by making site-directed mutants.     

Crystal structure of the kringle 2 domain of tissue plasminogen activator at 2.4-A resolution.

The crystal structure of the kringle 2 domain of tissue plasminogen activator was determined and refined at a resolution of 2.43 A. The overall fold of the molecule is similar to that of prothrombin kringle 1 and plasminogen kringle 4; however, there are differences in the lysine binding pocket, and two looping regions, which include insertions in kringle 2, take on very different conformations. Based on a comparison of the overall structural homology between kringle 2 and kringle 4, a new sequence alignment for kringle domains is proposed that results in a division of kringle domains into two groups, consistent with their proposed evolutionary relation. The crystal structure shows a strong interaction between a lysine residue of one molecule and the lysine/fibrin binding pocket of a noncrystallographically related neighbor. This interaction represents a good model of a bound protein ligand and is the first such ligand that has been observed in a kringle binding pocket. The structure shows an intricate network of interactions both among the binding pocket residues and between binding pocket residues and the lysine ligand. A lysine side chain is identified as the positively charged group positioned to interact with the carboxylate of lysine and lysine analogue ligands. In addition, a chloride ion is located in the kringle-kringle interface and contributes to the observed interaction between kringle molecules.     

Crystal structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin at 2.0 A resolution

1-Deoxynojirimycin, a pseudo-monosaccharide, is a strong inhibitor of glucoamylase but a relatively weak inhibitor of cyclodextrin glucanotransferase (CGTase). To elucidate this difference, the crystal structure of the CGTase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin was determined at 2.0 A resolution with the crystallographic R value of 0.154 (R(free) = 0.214). The asymmetric unit of the crystal contains two CGTase molecules and each molecule binds two 1-deoxynojirimycins. One 1-deoxynojirimycin molecule is bound to the active center by hydrogen bonds with catalytic residues and water molecules, but its binding mode differs from that expected in the substrate binding. Another 1-deoxynojirimycin found at the maltose-binding site 1 is bound to Asn-667 with a hydrogen bond and by stacking interaction with the indole moiety of Trp-662 of molecule 1 or Trp-616 of molecule 2. Comparison of this structure with that of the acarbose-CGTase complex suggested that the lack of stacking interaction with the aromatic side chain of Tyr-100 is responsible for the weak inhibition by 1-deoxynojirimycin of the enzymatic action of CGTase.     

Crystal structure of Ca2+ -free S100A2 at 1.6-A resolution

S100A2 is an EF hand-containing Ca^2 +-binding protein of the family of S100 proteins. The protein is localized exclusively in the nucleus and is involved in cell cycle regulation. It attracted most interest by its function as a tumor suppressor via p53 interaction. We determined the crystal structure of homodimeric S100A2 in the Ca^2 +-free state at 1.6-Å resolution. The structure revealed structural differences between subunits A and B, especially in the conformation of a loop that connects the N- and C-terminal EF hands and represents a part of the target-binding site in S100 proteins. Analysis of the hydrogen bonding network and molecular dynamics calculations indicate that one of the two observed conformations is more stable. The structure revealed Na⁺ bound to each N-terminal EF hand of both subunits coordinated by oxygen atoms of the backbone carbonyl and water molecules. Comparison with the structures of Ca^2 +-free S100A3 and S100A6 suggests that Na⁺ might occupy the S100-specific EF hand in the Ca^2 +-free state.     

Crystal structure of Escherichia coli CheY refined at 1.7-A resolution

The three-dimensional structure of wild-type CheY from Escherichia coli has been refined by stereochemically restrained least squares minimization to a crystallographic R-factor of 15.1% at 1.7-A resolution. The structure contains 1165 atoms, including all atoms of the protein, 147 water molecules, and three sulfate ions. The final model has root mean square deviations of 0.018 and 0.049 A from idealized bond lengths and angle distances, respectively. Seven amino acid side chains have been modeled in dual conformations. CheY folds as a compact (beta/alpha)5 globular protein, with the phosphorylation region contained in a cavity on one face of the molecule. This active site area is bordered by the carboxyl termini of the three central beta-strands, by alpha 1, and by the loop connecting beta 5 to alpha 5. The Lys-109 side chain of this loop extends into the active site by virtue of its cis peptide bond conformation preceding Pro-110. The epsilon-amino group of Lys-109 is in close bonding contact with the carboxyl group of Asp-57, the residue that is phosphorylated in the activation process of CheY. The details of the hydrogen bonding network in the phosphorylation region indicate that structural rearrangements must accompany the phosphorylation of Asp-57.