The x-ray crystal structure of phosphomannose isomerase from Candida albicans at 1.7 angstrom resolution

Phosphomannose isomerase (PMI) catalyses the reversible isomerization of fructose-6-phosphate (F6P) and mannose-6-phosphate (M6P). Absence of PMI activity in yeasts causes cell lysis and thus the enzyme is a potential target for inhibition and may be a route to antifungal drugs. The 1.7 A crystal structure of PMI from Candida albicans shows that the enzyme has three distinct domains. The active site lies in the central domain, contains a single essential zinc atom, and forms a deep, open cavity of suitable dimensions to contain M6P or F6P The central domain is flanked by a helical domain on one side and a jelly-roll like domain on the other.     

Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution

BACKGROUND: Barley alpha-amylase is a 45 kDa enzyme which is involved in starch degradation during barley seed germination. The released sugars provide the plant embryo with energy for growth. The major barley alpha-amylase isozyme (AMY2) binds with high affinity to the endogenous inhibitor BASI (barley alpha-amylase/subtilisin inhibitor) whereas the minor isozyme (AMY1) is not inhibited. BASI is a 19.6 kDa bifunctional protein that can simultaneously inhibit AMY2 and serine proteases of the subtilisin family. This inhibitor may therefore prevent degradation of the endosperm starch during premature sprouting and protect the seed from attack by pathogens secreting proteases. RESULTS: The crystal structure of AMY2 in complex with BASI was determined and refined at 1.9 A resolution. BASI consists of a 12-stranded beta-barrel structure which belongs to the beta-trefoil fold family and inhibits AMY2 by sterically occluding access of the substrate to the active site of the enzyme. The AMY2-BASI complex is characterized by an unusual completely solvated calcium ion located at the protein-protein interface. CONCLUSIONS: The AMY2-BASI complex represents the first reported structure of an endogenous protein-protein complex from a higher plant. The structure of the complex throws light on the strict specificity of BASI for AMY2, and shows that domain B of AMY2 contributes greatly to the specificity of enzyme-inhibitor recognition. In contrast to the three-dimensional structures of porcine pancreatic alpha-amylase in complex with proteinaceous inhibitors, the AMY2-BASI structure reveals that the catalytically essential amino acid residues of the enzyme are not directly bound to the inhibitor. Binding of BASI to AMY2 creates a cavity, exposed to the external medium, that is ideally shaped to accommodate an extra calcium ion. This feature may contribute to the inhibitory effect, as the key amino acid sidechains of the active site are in direct contact with water molecules which are in turn ligated to the calcium ion.     

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C-terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 A resolution. The structure reveals a compact fold distantly related to the C-type lectin carbohydrate recognition domain and the hyaluronan-binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.     

The crystal and molecular structure of trichosanthin at 2.6 A resolution

The crystallographic refinement of trichosanthin has been performed at 2.6 A resolution. The crystal and molecular structure of trichosanthin is described in detail in this paper. On summarizing the regularity of the amino acid sequences of eight kinds of ribosome inactivating proteins and combining with the crystal and molecular structure of trichosanthin, fifteen most conservative amino acid residues are analyzed. It is found that four most conservative polar amino acid residues Gln156, Glu160, Arg163 and Glu189 gather on the molecular surface on the boundary of the large and small domains, thus forming the active center of the protein molecule.     

The crystal structure of bacteriophage Q beta at 3.5 A resolution

BACKGROUND: The capsid protein subunits of small RNA bacteriophages form a T = 3 particle upon assembly and RNA encapsidation. Dimers of the capsid protein repress translation of the replicase gene product by binding to the ribosome binding site and this interaction is believed to initiate RNA encapsidation. We have determined the crystal structure of phage Q beta with the aim of clarifying which factors are the most important for particle assembly and RNA interaction in the small phages. RESULTS: The crystal structure of bacteriophage Q beta determined at 3.5 A resolution shows that the capsid is stabilized by disulfide bonds on each side of the flexible loops that are situated around the fivefold and quasi-sixfold axes. As in other small RNA phages, the protein capsid is constructed from subunits which associate into dimers. A contiguous ten-stranded antiparallel beta sheet facing the RNA is formed in the dimer. The disulfide bonds lock the constituent dimers of the capsid covalently in the T = 3 lattice. CONCLUSIONS: The unusual stability of the Q beta particle is due to the tight dimer interactions and the disulfide bonds linking each dimer covalently to the rest of the capsid. A comparison with the structure of the related phage MS2 shows that although the fold of the Q beta coat protein is very similar, the details of the protein-protein interactions are completely different. The most conserved region of the protein is at the surface, which, in MS2, is involved in RNA binding.     

The crystal structure of the processive endocellulase CelF of Clostridium cellulolyticum in complex with a thiooligosaccharide inhibitor at 2.0 A resolution

The mesophilic bacterium Clostridium cellulolyticum exports multienzyme complexes called cellulosomes to digest cellulose. One of the three major components of the cellulosome is the processive endocellulase CelF. The crystal structure of the catalytic domain of CelF in complex with two molecules of a thiooligosaccharide inhibitor was determined at 2.0 A resolution. This is the first three-dimensional structure to be solved of a member of the family 48 glycosyl hydrolases. The structure consists of an (alpha alpha)6-helix barrel with long loops on the N-terminal side of the inner helices, which form a tunnel, and an open cleft region covering one side of the barrel. One inhibitor molecule is enclosed in the tunnel, the other exposed in the open cleft. The active centre is located in a depression at the junction of the cleft and tunnel regions. Glu55 is the proposed proton donor in the cleavage reaction, while the corresponding base is proposed to be either Glu44 or Asp230. The orientation of the reducing ends of the inhibitor molecules together with the chain translation through the tunnel in the direction of the active centre indicates that CelF cleaves processively cellobiose from the reducing to the non-reducing end of the cellulose chain.     

X-ray crystal structure of a pea lectin-trimannoside complex at 2.6 A resolution

The x-ray crystal structure of pea lectin, in complex with a methyl glycoside of the N-linked-type oligosaccharide trimannosyl core, methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, has been solved by molecular replacement and refined at 2.6-A resolution. The R factor is 0.183 for all data in the 8.0 to 2.6 A resolution range with an average atomic temperature factor of 26.1 A2. Strong electron density for a single mannose residue is found in the monosaccharide-binding site suggesting that the trisaccharide binds primarily through one of the terminal alpha-linked mannose residues. The complex is stabilized by hydrogen bonds involving the protein residues Asp-81, Gly-99, Asn-125, Ala-217, and Glu-218, and the carbohydrate oxygen atoms O3, O4, O5, and O6. In addition, the carbohydrate makes van der Waals contacts with the protein, involving Phe-123 in particular. These interactions are very similar to those found in the monosaccharide complexes with concanavalin A and isolectin 1 of Lathyrus ochrus, confirming the structural relatedness of this family of proteins. Comparison of the pea lectin complex with the unliganded pea lectin and concanavalin A structures indicates differences in the conformation and water structure of the unliganded binding sites of these two proteins. Furthermore, a correlation between the position of the carbohydrate oxygen atoms in the complex and the bound water molecules in the unliganded binding sites is found. Binding of the trimannose core through a single terminal monosaccharide residue strongly argues that an additional fucose-binding site is responsible for the high affinity pea lectin-oligosaccharide interactions.     

X-ray structure at 1.76 A resolution of a polypeptide phospholipase A2 inhibitor

The high resolution crystal structure of a natural PLA2 inhibitor has been determined by Patterson search methods. In the heterodimeric, neurotoxic complex, vipoxin, isolated from the venom of Bulgarian viper, PLA2 inhibitor represents the non-toxic subunit. The model was refined to a crystallographic R-factor of 15.5% for data between 6 and 1.76 A resolution. The packing of the inhibitor in the crystal reveals close contacts between the molecules, which are symmetry-related by the 2-fold axes of the lattice. These pairs associate as a crystallographic dimer, stabilized by a set of interactions, including van der Waals contacts between residues from symmetry-related pairs, denoted as the recognition site and the recognition surface. Residues Ph3, Trp31 and Tyr119 represent the recognition site of inhibitor which possibly fits to the hydrophobic wall of the target PLA2. The topology of the inhibitor represents the PLA2 type of folding: three long helices and a beta-hairpin. Superposition of the structure of the inhibitor shows an almost complete overlap with different mammalian and viper PLA2 in the backbone and in the position of the sidechains of the residues that belong to the active centre and the hydrophobic wall. A "lock and key" mechanism of recognition of its native PLA2 in gland cells and other toxic PLA2 in vitro has been suggested. The mechanism includes complementary "head to tail" interactions between the recognition site of the inhibitor and a recognition surface located on the hydrophobic wall of the target PLA2. Having a high spatial homology with the PLA2 family of enzymes but opposing their action, the inhibitor from vipoxin presents an example of a divergent evolution of an ancient PLA2. The presence of a space for binding calcium in the inhibitor is believed to be a rudiment and proof of a common origin with PLA2.     

Refined crystal structure of the seryl-tRNA synthetase from Thermus thermophilus at 2.5 A resolution

The three-dimensional structure of the seryl-tRNA synthetase from Thermus thermophilus has been determined and refined at 2.5 A resolution. The final model consists of a dimer of 421 residues each and 190 water molecules. The R-factor is 18.4% for all the data between 10 and 2.5 A resolution. The structure is very similar to that of the homologous enzyme from Escherichia coli, with an r.m.s. difference of 1.5 A for the 357 alpha-carbon atoms considered equivalent. The comparison of the two structures indicates increased hydrophobicity, reduced conformational entropy and reduced torsional strain as possible mechanisms by which thermostability is obtained in the enzyme from the thermophile.     

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution

BACKGROUND: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. RESULTS: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G.A mismatches, are intercalated and stacked on top of each other without any interstrand Watson-Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion. CONCLUSIONS: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.     

Refined crystal structure of methylamine dehydrogenase from Paracoccus denitrificans at 1.75 A resolution

We extend the random intercept logistic model to accommodate negative intracluster correlations for bivariate binary response data. This approach assumes a single random effect per cluster, but entails separate affine transformations of this random effect for the two responses of the pair. We show this approach works for two data sets and a simulation, whereas other mixed effects approaches fail. The two data sets are from a crossover trial and a developmental toxicity study of the effects of chemical exposure on malformation risk among rat pups. Comparisons are made with the conditional likelihood approach and with generalized estimating equations estimation of the population-averaged logit model. Simulations show the conditional likelihood approach does not perform well for moderate to strong negative correlations, as a positive intracluster correlation is assumed. The proposed mixed effects approach appears to be slightly more conservative than the population-averaged approach with respect to coverage of confidence intervals. Nonetheless, the statistical literature suggests that mixed effects models provide information in addition to that provided by population-averaged models under scientific contexts such as crossover trials. Extensions to trivariate and higher-dimensional responses also are addressed. However, such extensions require certain constraints on the correlation structure.     

The crystal structure of seleno-glutathione peroxidase from human plasma at 2.9 A resolution

Glutathione peroxidase belongs to the family of selenoproteins and plays an important role in the defense mechanisms of mammals, birds and fish against oxidative damage by catalyzing the reduction of a variety of hydroperoxides, using glutathione as the reducing substrate. However, the physiological role of human plasma glutathione peroxidase remains unclear due to the low levels of reduced glutathione in human plasma and the low reactivity of this enzyme. The crystal structure of human plasma glutathione peroxidase was determined by Patterson search methods using a polyalanine model modified from the known structure of bovine erythrocyte glutathione peroxidase. The structure was refined to a crystallographic R-factor of 0.228 (R(free) = 0.335) with I > 2sigma(I) reflections in the resolution range of 8 to 2.9 A. The asymmetric unit contains a dimer. Tetramers are built up from dimers by crystallographic symmetry. The subunit structure of the plasma enzyme shows the typical structure motif of the thioredoxin fold consisting of a central beta-sheet and several flanking alpha-helices. The active site selenocysteine residue is situated in the loop between beta1 and alpha1 and is located in a pocket on the protein surface. The overall structure of the human plasma enzyme is similar to that of the bovine erythrocyte enzyme. The main differences in their subunit structures are an extended N terminus and the possible existence of a disulfide bridge in the plasma enzyme. Compared to the bovine erythrocyte enzyme, a number of residues in the active site are mutated or deleted in the plasma enzyme, including all the residues that were previously suggested to be involved in glutathione binding. The observed structural differences between the two enzymes suggest differences in substrate binding and specificity.     

Three-dimensional structure of human tissue inhibitor of metalloproteinases-2 at 2.1 A resolution

The three-dimensional structure of human tissue inhibitor of metalloproteinases-2 (TIMP-2) was determined by X-ray crystallography to 2.1 A resolution. The structure of the inhibitor consists of two domains. The N-terminal domain (residues 1-110) is folded into a beta-barrel, similar to the oligonucleotide/oligosaccharide binding fold otherwise found in certain DNA-binding proteins. The C-terminal domain (residues 111-194) contains a parallel stranded beta-hairpin plus a beta-loop-beta motif. Comparison of the structure of uncomplexed human TIMP-2 with that of bovine TIMP-2 bound to the catalytic domain of human MMP-14 suggests an internal rotation between the two domains of approximately 13 degrees upon binding to the protease. Furthermore, local conformational differences in the two structures that might be induced by formation of the protease-inhibitor complex have been found. The most prominent of these involves residues 27-40 of the A-B beta-hairpin loop. Structure-based alignment of amino acid sequences of representatives of the TIMP family maps the sequence differences mainly to loop regions, and some of these differences are proposed to be responsible for the particular properties of the various TIMP species.     

The crystal structure of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution

The emission spectrum of TaO, excited in a tantalum hollow cathode lamp, has been observed at high resolution using a Fourier transform spectrometer. In addition to previously known transitions, a number of new bands have been identified and assigned as belonging to two new electronic transitions: H2Pi1/2-X2Delta3/2 and K2Phi5/2-X2Delta3/2. A rotational analysis ofthe 0-0 and 0-1 bands of the H2Pi1/2-X2Delta3/2 transition and of the 0-1, 1-2, 0-0, 1-0, and 2-1 bands of theK2Phi5/2-X2Delta3/2 transition has been carried out, providing the following equilibrium constants for the ground X2Delta3/2 state:omegae = 1028.9060(15) cm-1, omegaexe = 3.58928(66) cm-1, Be = 0.40289737(139) cm-1, alphae = 0.00185445(83) cm-1, andre = 1.6873430(29) ?. The principal molecular constants for the H2Pi1/2 state are T00 = 20 634.32758 (40) cm-1,B0 = 0.3766867(31) cm-1, and r0 = 1.7450604(72) ?, while the equilibrium constants for the K2Phi5/2 state areomegae = 905.4549(15) cm-1, omegaexe = 3.67601(64) cm-1, Be = 0.37965102(36) cm-1, alphae = 0.00189370(21) cm-1, andre = 1.7382343(8) ?. Although the H2Pi1/2 and K2Phi5/2 states have been observed previously in matrix isolation experiments, our work on these states is the first in the gas phase. Copyright 1998 Academic Press.     

Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolpyruvate at 2.0 A resolution

Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes. Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP. The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively. In this structure, the dimer constitutes the crystallographic asymmetric unit. The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A. The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169. In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules. A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice. The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates. The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211. The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.     

The crystal structure of the sevenfold mutant of barley beta-amylase with increased thermostability at 2.5 A resolution

The three-dimensional structure of the sevenfold mutant of barley beta-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P43212 with cell dimensions a=b=72.11 A, c=250.51 A. The diffraction data up to 2.5 A were collected after soaking the crystal in 100 mM maltose with Rsym of 8.6 %. The structure was determined by a molecular replacement method using soybean beta-amylase (SBA) as a search model and refined to an R-factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 Calpha atoms between BBA-7s and SBA was 0.62 A. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent beta-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.     

Crystal structure at 1.95 A resolution of the breast tumour-specific antibody SM3 complexed with its peptide epitope reveals novel hypervariable loop recognition

The anti-breast tumour antibody SM3 has a high selectivity in reacting specifically with carcinoma-associated mucin. SM3 recognises the core repeating motif (Pro-Asp-Thr-Arg-Pro) of aberrantly glycosylated epithelial mucin MUC1, and has potential as a therapeutic and diagnostic tool. Here we report the crystal structure of the Fab fragment of SM3 in complex with a 13-residue MUC1 peptide antigen (Thr1P-Ser2P-Ala3P-Pro4P-Asp5P-Thr6P -Arg7P-Pro8P-Ala9P-Pro10P-Gly11P- Ser12P-Thr13P). The SM3-MUC1 peptide structure was solved by molecular replacement, and the current model is refined at 1.95 A resolution with an R-factor of 21.3% and R-free 28.3%. The MUC1 peptide is bound both by non-polar interactions and hydrogen bonds in an elongated groove in the antibody-combining site through interactions with Complimentarity Determining Regions (CDRs), three of the light chain (L1, L2, L3) and two of the heavy chain (H1 and H3). The conformation of the peptide is mainly extended with no discernable standard secondary structure. There is a single non-proline cis-peptide bond in H3 (Val95H-Gly96H-Gln97H-Phe98H-Ala101H-Ty r102H) between Gly96H and Gln97H, which appears to play a role in SM3-peptide antigen interactions, and represents the first such example within an antibody hypervariable loop. The SM3-MUC1 peptide structure has implications for rational therapeutic and diagnostic antibody engineering.     

Refined crystal structure of a superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius at 2.2 A resolution

The extremely thermostable superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius was crystallized and the three-dimensional structure was determined by X-ray diffraction methods. The enzyme crystallized in the monoclinic spacegroup C2 with the cell dimensions a=168.1 A, b=91.3 A, c=85.7 A, beta=91.4 degrees. The diffraction limit of these crystals was 2.2 A. The crystals were very stable in the X-ray beam and measured diffraction data of a single crystal had a completeness of 99.5 % up to a resolution of 2.2 A. The crystal structure of S. acidocaldarius superoxide dismutase was solved by Patterson search methods using a dimer of Thermus thermophilus superoxide dismutase as a search model. The asymmetric unit accommodates three dimers. Two dimers form a tetramer by using only local symmetries; the third dimer forms a tetramer as well, however, by using the crystallographic 2-fold symmetry. The three-dimensional structure of the S. acidocaldarius dismutase has typical features of tetrameric dismutases. Secondary structure elements as well as residues important for the catalytic activity of the enzyme were found to be highly conserved. The model was refined at a resolution of 2.2 A and yielded a crystallographic R-value of 17.4 % (Rfree=22.3 %). A structural comparison of the two extremely stable tetrameric dismutases from S. acidocaldarius and Aquifex pyrophilus with the less stable enzyme from T. thermophilus and Mycoplasma tuberculosis revealed the structural determinants which are probably responsible for the high intrinsic stability of S. acidocaldarius dismutase. The most obvious factor which may give rise to the extraordinary thermal stability of S. acidocaldarius dismutase (melting temperature of about 125 degreesC) is the increase in intersubunit ion pairs and hydrogen bonds and, more importantly, the significant reduction of solvent-accessible hydrophobic surfaces, as well as an increase in the percentage of buried hydrophobic residues.     

The crystal structure of an Fe-superoxide dismutase from the hyperthermophile Aquifex pyrophilus at 1.9 A resolution: structural basis for thermostability

The incidence of stress fractures is increasing among competitive and recreational athletes as well as among children and the elderly. By understanding the continuum of bone's response to stress and maintaining an appropriate index of suspicion, the health care provider can diagnose these injuries appropriately. An accurate history and examination is essential and will differentiate stress fractures from other stress reactions. The more common stress fractures are discussed.