The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4.

The X-ray crystal structure of an anti-leukotriene (LT) C4 monoclonal antibody (mAbLTC) in complex with LTC4 was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC4, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC4 single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE4 which was comparable to that to LTD4 Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE4 The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC4 was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies.

BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.

Functional expression of single-chain antibody to leukotriene C(4).

Leukotriene C(4) (LTC(4)) is synthesized by binding of glutathione to LTA(4), an epoxide derived from arachidonic acid, and further metabolized to LTD(4) and LTE(4). We previously prepared a monoclonal antibody with a high affinity and specificity to LTC(4). To explore the structure of the antigen-binding site of a monoclonal antibody against LTC(4) (mAbLTC), we isolated full-length cDNAs for heavy and light chains of mAbLTC. The heavy and light chains consisted of 461 and 238 amino acids including a signal peptide with molecular weights of 51,089 and 26,340, respectively. An expression plasmid encoding a single-chain antibody comprising variable regions of mAbLTC heavy and light chains (scFvLTC) was constructed and expressed in COS-7 cells. The recombinant scFvLTC showed a high affinity with LTC(4) comparable to mAbLTC. The scFvLTC also bound to LTD(4) and LTE(4) with 48% and 17% reactivities, respectively, as compared with LTC(4) binding, whereas the antibody showed almost no affinity for LTB(4).

Crystallization and preliminary diffraction studies of prostaglandin E2-specific monoclonal antibody Fab fragment in the ligand complex.

Prostaglandin E(2) is a major lipid mediator that regulates diverse biological processes. To elucidate how prostaglandin E(2) is recognized specifically by its antibody, the Fab fragment of a monoclonal anti-prostaglandin E(2) antibody was prepared and its complex with prostaglandin E(2) was crystallized. The stable Fab-prostaglandin E(2) complex was prepared by gel-filtration chromatography. Crystals were obtained by the microbatch method at 277 K using polyethylene glycol 4000 as a precipitant. A diffraction data set was collected to 2.2 A resolution. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.3, b = 81.8, c = 82.2 A. The asymmetric unit was suggested to contain one molecule of the Fab-prostaglandin E(2) complex, with a corresponding crystal volume per protein weight of 2.75 A(3) Da(-1).

Structure of a haloacid dehalogenase superfamily phosphatase PH1421 from Pyrococcus horikoshii OT3: oligomeric state and thermoadaptation mechanism.

PH1421 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is a hypothetical protein belonging to the haloacid dehalogenase (HAD) superfamily. To gain insight into its biological function and thermostabilization mechanism, the crystal structure of PH1421 has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a homodimer. The monomeric protomer is composed of two distinct domains, a small cap domain and a large core domain, which agrees well with the typical domain organization of HAD subfamily II. Based on structure-based amino-acid sequence alignment and enzymatic analysis, PH1421 is suggested to be a magnesium-dependent phosphatase that is similar to the dimeric HAD phosphatase TA0175 from the mesothermophilic archaeon Thermoplasma acidophilum. Further comparison between the crystal structures of PH1421 and TA0175 revealed a marked structural similarity in the interprotomer dimer association. The common dimer interface with interprotomer twofold symmetry is characterized by a well conserved hydrophobic core consisting of the beta1-alpha1 loop and helices alpha1 and alpha2 of the core domain and additional contacts including the beta7-beta8 loop of the cap domain, which constitutes part of the putative active site of the enzyme. Several factors that potentially contribute to the higher thermal stability of PH1421 were identified: (i) an increase in intraprotomer hydrophobic interactions, (ii) a decrease in denaturation entropy from amino-acid composition and (iii) an increased number of intraprotomer ion pairs. These results suggest that the PH1421 protomer itself has an intrinsically higher thermal stability when compared with the mesothermophilic orthologue TA0175.

High-throughput crystallization-to-structure pipeline at RIKEN SPring-8 Center.

A high-throughput crystallization-to-structure pipeline for structural genomics was recently developed at the Advanced Protein Crystallography Research Group of the RIKEN SPring-8 Center in Japan. The structure determination pipeline includes three newly developed technologies for automating X-ray protein crystallography: the automated crystallization and observation robot system "TERA", the SPring-8 Precise Automatic Cryosample Exchanger "SPACE" for automated data collection, and the Package of Expert Researcher's Operation Network "PERON" for automated crystallographic computation from phasing to model checking. During the 5 years following April, 2002, this pipeline was used by seven researchers to determine 138 independent crystal structures (resulting from 437 purified proteins, 234 cryoloop-mountable crystals, and 175 diffraction data sets). The protocols used in the high-throughput pipeline are described in this paper.

Crystallization screening test for the whole-cell project on Thermus thermophilus HB8.

It was essential for the structural genomics of Thermus thermophilus HB8 to efficiently crystallize a number of proteins. To this end, three conventional robots, an HTS-80 (sitting-drop vapour diffusion), a Crystal Finder (hanging-drop vapour diffusion) and a TERA (modified microbatch) robot, were subjected to a crystallization condition screening test involving 18 proteins from T. thermophilus HB8. In addition, a TOPAZ (microfluidic free-interface diffusion) designed specifically for initial screening was also briefly examined. The number of diffraction-quality crystals and the time of appearance of crystals increased in the order HTS-80, Crystal Finder, TERA. With the HTS-80 and Crystal Finder, the time of appearance was short and the rate of salt crystallization was low. With the TERA, the number of diffraction-quality crystals was high, while the time of appearance was long and the rate of salt crystallization was relatively high. For the protein samples exhibiting low crystallization success rates, there were few crystallization conditions that were common to the robots used. In some cases, the success rate depended greatly on the robot used. The TOPAZ showed the shortest time of appearance and the highest success rate, although the crystals obtained were too small for diffraction studies. These results showed that the combined use of different robots significantly increases the chance of obtaining crystals, especially for proteins exhibiting low crystallization success rates. The structures of 360 of 944 purified proteins have been successfully determined through the combined use of an HTS-80 and a TERA.

Structure of peptidyl-tRNA hydrolase 2 from Pyrococcus horikoshii OT3: insight into the functional role of its dimeric state.

Peptidyl-tRNA hydrolases catalyze the hydrolytic removal of the peptidyl moiety from the peptidyl-tRNA molecule to prevent misreading during translation. Here, the expression, purification, crystallization and X-ray diffraction study of peptidyl-tRNA hydrolase 2 from Pyrococcus horikoshii OT3 (PhPth2) are described. The crystal structures were determined as similar biological dimers in two different forms: P4(1)2(1)2 at 1.2 A resolution (form 1) and P4(3)22 at 1.9 A resolution (form 2). In the form 1 structure, the asymmetric unit contains one PhPth2 subunit and a crystallographic twofold axis defines the dimeric association with the cognate subunit. In the form 2 structure, there are two PhPth2 subunits in the asymmetric unit that make a similar dimer with a noncrystallographic twofold axis. In order to evaluate the thermodynamic stability, the intra-protomer and inter-protomer interactions of PhPth2 were analyzed and compared with those of other Pth2-family members. The thermodynamic parameters show that the large number of ion pairs compared with family members from other mesophilic organisms would contribute to the thermostability of PhPth2. The structural difference between the two dimers was quantitatively evaluated by a multiple C(alpha)-atom superposition. A significant structural difference between the two dimers was observed around the putative active site of this enzyme. A rigid-body rotation takes place so as to retain the dimeric twofold symmetry, suggesting positive cooperativity upon tRNA binding. The mechanism of ligand binding was further investigated using a docking model with a tRNA molecule. The docking study suggests that the binding of tRNA requires its simultaneous interaction with both subunits of the PhPth2 dimer.

Purification, crystallization and preliminary crystallographic analysis of archaeal 6-pyruvoyl tetrahydrobiopterin synthase homologue PH0634 from Pyrococcus horikoshii OT3.

6-Pyruvoyl tetrahydrobiopterin synthase (PTPS) catalyses the conversion of dihydroneopterin triphosphate to 6-pyruvoyl tetrahydropterin, the second of the three enzymatic steps in the synthesis of tetrahydrobiopterin from GTP. PH0634, a 13.51 kDa archaeal PTPS homologue from Pyrococcus horikoshii OT3, was overexpressed as native and selenomethionine-substituted protein and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data were collected to 2.1 A resolution from the native crystal using synchrotron radiation at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 35.83, b = 95.71, c = 105.65 A. Threefold noncrystallographic symmetry was identified from self-rotation calculations. Assuming the presence of a trimer in the asymmetric unit, the solvent content is 45% (V(M) = 2.24 A3 Da(-1)). The selenomethionine-substituted crystal is isomorphous to the native crystal and diffracts X-rays to 2.9 A.

Purification, crystallization and preliminary crystallographic analysis of the putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3.

The putative thiamine-biosynthesis protein PH1313 from Pyrococcus horikoshii OT3 has been overexpressed and purified. Crystallization was performed by the oil-microbatch method using 28%(v/v) 2-methyl-2,4-pentanediol as a precipitant at 291 K. A native X-ray diffraction data set at 1.9 A resolution and a single anomalous dispersion data set from a selenomethionine-derivative crystal at 2.1 A resolution were collected using synchrotron radiation at 100 K. The native crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 71.7, b = 71.2, c = 141.8 A.

Integrated state evaluation for the images of crystallization droplets utilizing linear and nonlinear classifiers.

In a usual crystallization process, the researchers evaluate the protein crystallization growth states based on visual impressions and repeatedly assign scores throughout the growth process. Although the development of crystallization robotic systems has generally realised the automation of the setup and storage of crystallization samples, evaluation of crystallization states has not yet been completely automated. The method presented here attempts to categorize individual crystallization droplet images into five classes using multiple classifiers. In particular, linear and nonlinear classifiers are utilized. The algorithm is comprised of pre-processing, feature extraction from images using texture analysis and a categorization process using linear discriminant analysis (LDA) and support vector machine (SVM). The performance of this method has been evaluated by comparing the results obtained using the method with the results obtained by a human expert and the concordance rate was 84.4%.

Evaluation of crystalline objects in crystallizing protein droplets based on line-segment information in greyscale images.

Several automated crystallization systems have recently been developed for high-throughput X-ray structure analysis. However, the evaluation process for the growth state of crystallizing protein droplets has not yet been completely automated. This paper proposes a new evaluation method for crystalline objects in automated crystallization experiments. The main objective is to determine whether a droplet contains crystals suitable for diffraction experiments and analysis. The evaluation method developed here involves extracting line-segment features from an image of the droplet and discriminating the state of crystallization using classifiers based on line features. In order to verify the efficacy of the proposed method, it was used to classify images obtained by an automated crystallization system.

Stabilization mechanism of the tryptophan synthase alpha-subunit from Thermus thermophilus HB8: X-ray crystallographic analysis and calorimetry.

In order to elucidate the thermo-stabilization mechanism of the tryptophan synthase alpha-subunit from the extreme thermophile Thermus thermophilus HB8 (Tt-alpha-subunit), its crystal structure was determined and its stability was examined using DSC. The results were compared to those of other orthologs from mesophilic and hyperthermophilic organisms. The denaturation temperature of the Tt-alpha-subunit was higher than that of the alpha-subunit from S. typhimurium (St-alpha-subunit) but lower than that of the alpha-subunit from P. furiosus (Pf-alpha-subunit). Specific denaturation enthalpy and specific denaturation heat capacity values of the Tt-alpha-subunit were the lowest among the three proteins, suggesting that entropy effects are responsible for the stabilization of the Tt-alpha-subunit. Based on a structural comparison with the St-alpha-subunit, two deletions in loop regions, an increase in the number of ion pairs and a decrease in cavity volume seem to be responsible for the stabilization of the Tt-alpha-subunit. The results of structural comparison suggest that the native structure of the Tt-alpha-subunit is better adapted to an ideally stable structure than that of the St-alpha-subunit, but worse than that of the Pf-alpha-subunit. The results of calorimetry suggest that the residual structure of the Tt-alpha-subunit in the denatured state contributes to the stabilization.

Structure of ATP-dependent phosphoenolpyruvate carboxykinase from Thermus thermophilus HB8 showing the structural basis of induced fit and thermostability.

In order to understand the induced fit and the thermostabilization mechanisms of ATP-dependent phosphoenolpyruvate carboxykinase, the crystal structure of the enzyme from the extreme thermophile Thermus thermophilus HB8 (TtPEPCK) was determined and compared with those of orthologues of known structure from two mesophilic organisms. The protomer structures in these orthologues, which exhibit open/closed interdomain conformations, are similar. Isomorphous crystals of unliganded and ATP-bound TtPEPCK were obtained. The asymmetric units of both crystal forms contain two protomers A and B with closed and open conformations, respectively. ATP was only observed in the interdomain cleft of the closed protomer, suggesting that the induced fit of TtPEPCK agrees with the so-called ;conformational selection' mechanism where ligand binding is not essential for domain closure although its binding leads to the stabilization of the closed state. A bound calcium observed in the N-terminal domain of TtPEPCK probably contributes to the thermal stability. A combination of hydrophobic effects, ion pairs and entropic effects might also contribute to the thermostability of TtPEPCK.

Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation.

Biotin protein ligase (EC 6.3.4.15) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. The three-dimensional structure of biotin protein ligase from Pyrococcus horikoshii OT3 has been determined by X-ray diffraction at 1.6A resolution. The structure reveals a homodimer as the functional unit. Each subunit contains two domains, a larger N-terminal catalytic domain and a smaller C-terminal domain. The structural feature of the active site has been studied by determination of the crystal structures of complexes of the enzyme with biotin, ADP and the reaction intermediate biotinyl-5'-AMP at atomic resolution. This is the first report of the liganded structures of biotin protein ligase with nucleotide and biotinyl-5'-AMP. The structures of the unliganded and the liganded forms are isomorphous except for an ordering of the active site loop upon ligand binding. Catalytic binding sites are suitably arranged to minimize the conformational changes required during the reaction, as the pockets for biotin and nucleotide are located spatially adjacent to each other in a cleft of the catalytic domain and the pocket for biotinyl-5'-AMP binding mimics the combination of those of the substrates. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by biotin protein ligase in which the positively charged epsilon-amino group of Lys111 facilitates the nucleophilic attack on the ATP alpha-phosphate group by the biotin carboxyl oxygen atom and stabilizes the negatively charged intermediates.

A novel induced-fit reaction mechanism of asymmetric hot dog thioesterase PAAI.

Hot dog fold proteins sharing the characteristic "hot dog" fold are known to involve certain coenzyme A binding enzymes with various oligomeric states. In order to elucidate the oligomerization-function relationship of the hot dog fold proteins, crystal structures of the phenylacetate degradation protein PaaI from Thermus thermophilus HB8 (TtPaaI), a tetrameric acyl-CoA thioesterase with the hot dog fold, have been determined and compared with those of other family members. In the liganded crystal forms with coenzyme A derivatives, only two of four intersubunit catalytic pockets of the TtPaaI tetramer are occupied by the ligands. A detailed structural comparison between several liganded and unliganded forms reveals that a subtle rigid-body rearrangement of subunits within 2 degrees upon binding of the first two ligand molecules can induce a strict negative cooperativity to prevent further binding at the remaining two pockets, indicating that the so-called "half-of-the-sites reactivity" of oligomeric enzymes is visualized for the first time. Considering kinetic and mutational analyses together, a possible reaction mechanism of TtPaaI is proposed; one tetramer binds only two acyl-CoA molecules with a novel asymmetric induced-fit mechanism and carries out the hydrolysis according to a base-catalyzed reaction through activation of a water molecule by Asp48. From a structural comparison with other family members, it is concluded that a subgroup of the hot dog fold protein family, referred to as "asymmetric hot dog thioesterases" including medium chain acyl-CoA thioesterase II from Escherichia coli and human thioesterase III, might share the same oligomerization mode and the asymmetric induced-fit mechanism as observed in TtPaaI.

Evaluation of protein crystallization states based on texture information derived from greyscale images.

In recent years, several projects have advanced research and development related to the automation of the protein crystallization process. However, evaluation of crystallization states has not yet been completely automated. In the usual crystallization process, researchers evaluate the protein crystallization growth states based on visual impressions and assign them a score over and over again. The method presented here automates this evaluation process. This method attempts to categorize the individual crystallization droplet images into five classes. The algorithm is comprised of pre-processing, feature extraction from images using texture analysis and a categorization process using linear discriminant analysis. The performance of this method has been evaluated by comparing the results obtained by using this method with the results from a human expert and the concordance rate was 90.6%.

Stabilization due to dimer formation of phosphoribosyl anthranilate isomerase from Thermus thermophilus HB8: X-ray Analysis and DSC experiments.

The crystal structure of phosphoribosyl anthranilate isomerase (PRAI) from Thermus thermophilus HB8 (TtPRAI) was solved at 2.0 A resolution. The overall structure of TtPRAI with a dimeric structure was quite similar to that of PRAI from Thermotoga maritima (TmPRAI). In order to elucidate the stabilization mechanism of TtPRAI, its physicochemical properties were examined using DSC, CD, and analytical centrifugation at various pHs in relation to the association-dissociation of the subunits. Based on the experimental results for TtPRAI and the structural information on TtPRAI and TmPRAI, we found that: (i) the denaturation of TtPRAI at acidic pH is correlated with the dissociation of its dimeric form; (ii) the hydrophobic interaction of TtPRAI in the monomer structure is slightly greater than that of TmPRAI, but dimer interface of the TmPRAI is remarkably greater; (iii) the contributions of hydrogen bonds and ion bonds to the stability are similar to each other; and (iv) destabilization due to the presence of cavities in TtPRAI is greater than that of TmPRAI in both the monomer and dimer structures.

Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3.

Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.

Purification, crystallization and initial X-ray crystallographic analysis of the putative GTPase PH0525 from Pyrococcus horikoshii OT3.

GTPases are involved in diverse cellular functions including cell proliferation, cytoskeleton organization and intracellular traffic. The putative GTPase PH0525 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Two distinct crystal forms were grown by the microbatch method at 291 K using a very high protein concentration (80 mg ml(-1)). Native data sets extending to resolutions of 2.3 and 2.4 A have been collected and processed in space groups P2(1) and C222(1), respectively. Assuming the presence of one monomer per asymmetric unit gives VM values of 2.6 and 2.4 A3 Da(-1) for the P2(1) and C222(1) forms, respectively, which is consistent with dynamic light-scattering experiments, which show a monomeric state of the protein in solution.