Identification of novel interacting regions involving calcineurin and nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) leads to the transcription of diverse inducible genes involved in many biological processes; therefore, aberrant NFAT expression is responsible for the development and exacerbation of various disorders. Since five isoforms of NFAT (NFATc1-c4, NFAT5) exhibit distinct and overlapping functions, selective control of a part, but not all, of NFAT family members is desirable. By comparing the binding activity of each NFATc1-c4 with its regulatory enzyme, calcineurin (CN), using a quantitative immunoprecipitation assay, we found a new CN-binding region (CNBR) selectively functioning in NFATc1 and NFATc4. This region, termed CNBR3, is located between two preexisting CNBR1 and CNBR2, within the Ca2+ regulatory domain. The nuclear translocation of NFATc1 but not NFATc2 in T cells was suppressed by ectopic expression of CNBR3 and, accordingly, NFATc1-dependent cytokine expression was downregulated. Through competition assays using NFATc1-derived partial peptides and mass spectrometry with photoaffinity technology, we identified 18 amino acids in NFATc1 (Arg258 to Pro275 ) and 13 amino acids in CN catalytic subunit (CNA) (Asn77 to Gly89 ) responsible for CNA/CNBR3 binding in which Cys263 and Asp82 , respectively, played crucial roles. The possible selective regulation of NFAT-mediated biological processes by targeting this new CN/NFAT-binding region is suggested.

Structural basis of different substrate preferences of two old yellow enzymes from yeasts in the asymmetric reduction of enone compounds.

Old yellow enzymes (OYEs) are potential targets of protein engineering for useful biocatalysts because of their excellent asymmetric reductions of enone compounds. Two OYEs from different yeast strains, Candida macedoniensis AKU4588 OYE (CmOYE) and Pichia sp. AKU4542 OYE (PsOYE), have a sequence identity of 46%, but show different substrate preferences; PsOYE shows 3.4-fold and 39-fold higher catalytic activities than CmOYE toward ketoisophorone and (4S)-phorenol, respectively. To gain insights into structural basis of their different substrate preferences, we have solved a crystal structure of PsOYE, and compared its catalytic site structure with that of CmOYE, revealing the catalytic pocket of PsOYE is wider than that of CmOYE due to different positions of Phe246 (PsOYE)/Phe250 (CmOYE) in static Loop 5. This study shows a significance of 3D structural information to explain the different substrate preferences of yeast OYEs which cannot be understood from their amino acid sequences. Abbreviations: OYE: Old yellow enzymes, CmOYE: Candida macedoniensis AKU4588 OYE, PsOYE: Pichia sp. AKU4542 OYE.

A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability.

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1-16h in the presence of 0-2.0% (w/v) n-dodecyl-ß-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.

REFOLDdb: a new and sustainable gateway to experimental protocols for protein refolding.

BACKGROUND: More than 7000 papers related to "protein refolding" have been published to date, with approximately 300 reports each year during the last decade. Whilst some of these papers provide experimental protocols for protein refolding, a survey in the structural life science communities showed a necessity for a comprehensive database for refolding techniques. We therefore have developed a new resource - "REFOLDdb" that collects refolding techniques into a single, searchable repository to help researchers develop refolding protocols for proteins of interest. RESULTS: We based our resource on the existing REFOLD database, which has not been updated since 2009. We redesigned the data format to be more concise, allowing consistent representations among data entries compared with the original REFOLD database. The remodeled data architecture enhances the search efficiency and improves the sustainability of the database. After an exhaustive literature search we added experimental refolding protocols from reports published 2009 to early 2017. In addition to this new data, we fully converted and integrated existing REFOLD data into our new resource. REFOLDdb contains 1877 entries as of March 17th, 2017, and is freely available at http://p4d-info.nig.ac.jp/refolddb/ . CONCLUSION: REFOLDdb is a unique database for the life sciences research community, providing annotated information for designing new refolding protocols and customizing existing methodologies. We envisage that this resource will find wide utility across broad disciplines that rely on the production of pure, active, recombinant proteins. Furthermore, the database also provides a useful overview of the recent trends and statistics in refolding technology development.

An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.

(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.

Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A2 from Neurospora crassa.

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.

Structural basis for the Ca(2+)-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190.

A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an α/ß hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca(2+)-induced thermostabilization and activation of enzymes have been well explored in α-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca(2+) enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190(S226P)) in the Ca(2+)-bound and free states at 1.75 and 1.45 Å resolution, respectively. Based on the crystallographic data, a Ca(2+) ion was coordinated by four residues within loop regions (the Ca(2+) site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca(2+) to Cut190(S226P) induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca(2+) not only stabilized a region that is flexible in the Ca(2+)-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca(2+)-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190(S226P) is activated by a conformational change in the active-site sealing residue, F106.

Structural units important for activity of a novel-type phosphoserine phosphatase from Hydrogenobacter thermophilus TK-6 revealed by crystal structure analysis.

Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent phosphoglycerate mutase (dPGM)-like phosphatases that have significant amino acid sequence similarity to dPGMs but lack phosphoglycerate mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate L-phosphoserine to find structural units important for its phosphatase activity toward L-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His(85) and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases.

Complex structure of the DNA-binding domain of AdpA, the global transcription factor in Streptomyces griseus, and a target duplex DNA reveals the structural basis of its tolerant DNA sequence specificity.

AdpA serves as the global transcription factor in the A-factor regulatory cascade, controlling the secondary metabolism and morphological differentiation of the filamentous bacterium Streptomyces griseus. AdpA binds to over 500 operator regions with the consensus sequence 5'-TGGCSNGWWY-3' (where S is G or C, W is A or T, Y is T or C, and N is any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To elucidate the structural basis of this tolerant DNA recognition by AdpA, we focused on the interaction between the DNA-binding domain of AdpA (AdpA-DBD), which consists of two helix-turn-helix motifs, and a target duplex DNA containing the consensus sequence 5'-TGGCGGGTTC-3'. The crystal structure of the AdpA-DBD-DNA complex and the mutant analysis of AdpA-DBD revealed its unique manner of DNA recognition, whereby only two arginine residues directly recognize the consensus sequence, explaining the strict recognition of G and C at positions 2 and 4, respectively, and the tolerant recognition of other positions of the consensus sequence. AdpA-DBD confers tolerant DNA sequence specificity to AdpA, allowing it to control hundreds of genes as a global transcription factor.

A novel mode of ferric ion coordination by the periplasmic ferric ion-binding subunit FbpA of an ABC-type iron transporter from Thermus thermophilus HB8.

Crystal structures of FbpA, the periplasmic ferric ion-binding protein of an iron-uptake ABC transporter, from Thermus thermophilus HB8 (TtFbpA) have been solved in apo and ferric ion-bound forms at 1.8 and 1.7 Šresolution, respectively. The latter crystal structure shows that the bound ferric ion forms a novel six-coordinated complex with three tyrosine side chains, two bicarbonates and a water molecule in the metal-binding site. The results of gel-filtration chromatography and dynamic light scattering show that TtFbpA exists as a monomer in solution regardless of ferric ion binding and that TtFbpA adopts a more compact conformation in the ferric ion-bound state than in the apo state in solution.

L-allo-threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity.

L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Šresolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Šoutwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

Human CTP:phosphoethanolamine cytidylyltransferase: enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains.

CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis-Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.

Crystal structure of the novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 reveals a special halide-stabilizing pair and enantioselectivity mechanism.

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. Because DatA possesses a unique Asn-Tyr pair instead of the Asn-Trp pair conserved among the subfamily members, which was proposed to keep the released halide ion stable, the structural basis for its reaction mechanism should be elucidated. Here, we determined the crystal structures of DatA and its Y109W mutant at 1.70 and 1.95 Å, respectively, and confirmed the location of the active site by using its novel competitive inhibitor. The structural information from these two crystal structures and the docking simulation suggested that (i) the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for some halogenated compounds, such as 1,3-dibromopropane, mainly due to the electrostatic interaction between Trp109 and halogenated compounds and the change of substrate-binding mode caused by the interaction and (ii) the primary halide-stabilizing residue is only Asn43 in the wild-type DatA, while Tyr109 is a secondary halide-stabilizing residue. Furthermore, docking simulation using the crystal structures of DatA indicated that its enantioselectivity is determined by the large and small spaces around the halogen-binding site.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

Crystal structure and site-directed mutagenesis analyses of haloalkane dehalogenase LinB from Sphingobium sp. strain MI1205.

The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of ß-hexachlorocyclohexane (ß-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from ß-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.

Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily.

High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7.

Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.

Crystallization and preliminary X-ray diffraction analysis of a novel type of phosphoserine phosphatase from Hydrogenobacter thermophilus TK-6.

Two novel-type phosphoserine phosphatases (PSPs) with unique substrate specificity from the thermophilic and hydrogen-oxidizing bacterium Hydrogenobacter thermophilus TK-6 have previously been identified. Here, one of the PSPs (iPSP1) was heterologously expressed in Escherichia coli, purified and crystallized. Diffraction-quality crystals were obtained by the sitting-drop vapour-diffusion method using PEG 4000 as the precipitant. Two diffraction data sets with resolution ranges of 45.0-2.50 and 45.0-1.50 Šwere collected from a single crystal and were merged to give a highly complete data set. The space group of the crystal was identified as primitive orthorhombic P2(1)2(1)2(1), with unit-cell parameters a = 49.8, b = 73.6, c = 124.3 Å. The calculated Matthews coefficient (V(M) = 2.32 Å(3) Da(-1)) indicated that the crystal contained one iPSP1 complex per asymmetric unit.

Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58.

Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit.

Purification, crystallization and preliminary X-ray analysis of the DNA-binding domain of AdpA, the central transcription factor in the A-factor regulatory cascade in the filamentous bacterium Streptomyces griseus, in complex with a duplex DNA.

Streptomyces griseus AdpA is the central transcription factor in the A-factor regulatory cascade and activates a number of genes that are required for both secondary metabolism and morphological differentiation, leading to the onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. The DNA-binding domain of AdpA consists of two helix-turn-helix DNA-binding motifs and shows low nucleotide-sequence specificity. To reveal the molecular basis of the low nucleotide-sequence specificity, an attempt was made to obtain cocrystals of the DNA-binding domain of AdpA and several kinds of duplex DNA. The best diffracting crystal was obtained using a 14-mer duplex DNA with two-nucleotide overhangs at the 5'-ends. The crystal diffracted X-rays to 2.8 Šresolution and belonged to space group C222(1), with unit-cell parameters a = 76.86, b = 100.96, c = 101.25 Å. The Matthews coefficient (V(M) = 3.71 Å(3) Da(-1)) indicated that the crystal was most likely to contain one DNA-binding domain of AdpA and one duplex DNA in the asymmetric unit, with a solvent content of 66.8%.