Structure of mitogen-activated protein kinase kinase 1 in the DFG-out conformation.

Eukaryotic protein kinases contain an Asp-Phe-Gly (DFG) motif, the conformation of which is involved in controlling the catalytic activity, at the N-terminus of the activation segment. The motif can be switched between active-state (DFG-in) and inactive-state (DFG-out) conformations: however, the mechanism of conformational change is poorly understood, partly because there are few reports of the DFG-out conformation. Here, a novel crystal structure of nonphosphorylated human mitogen-activated protein kinase kinase 1 (MEK1; amino acids 38-381) complexed with ATP-γS is reported in which MEK1 adopts the DFG-out conformation. The crystal structure revealed that the structural elements (the αC helix and HRD motif) surrounding the active site are involved in the formation/stabilization of the DFG-out conformation. The ATP-γS molecule was bound to the canonical ATP-binding site in a different binding mode that has never been found in previously determined crystal structures of MEK1. This novel ATP-γS binding mode provides a starting point for the design of high-affinity inhibitors of nonphosphorylated inactive MEK1 that adopts the DFG-out conformation.

Structures of endo-1,5-α-L-arabinanase mutants from Bacillus thermodenitrificans TS-3 in complex with arabino-oligosaccharides.

The thermostable endo-1,5-α-L-arabinanase from Bacillus thermodenitrificans TS-3 (ABN-TS) hydrolyzes the α-1,5-L-arabinofuranoside linkages of arabinan. In this study, the crystal structures of inactive ABN-TS mutants, D27A and D147N, were determined in complex with arabino-oligosaccharides. The crystal structures revealed that ABN-TS has at least six subsites in the deep V-shaped cleft formed across one face of the propeller structure. The structural features indicate that substrate recognition is profoundly influenced by the remote subsites as well as by the subsites surrounding the active center. The `open' structure of the substrate-binding cleft of the endo-acting ABN-TS is suitable for the random binding of several sugar units in polymeric substrates.

Crystal structure of exo-rhamnogalacturonan lyase from Penicillium chrysogenum as a member of polysaccharide lyase family 26.

Exo-rhamnogalacturonan lyase from Penicillium chrysogenum 31B (PcRGLX) was recently classified as a member of polysaccharide lyase (PL) family 26 along with hypothetical proteins derived from various organisms. In this study, we determined the crystal structure of PcRGLX as the first structure of a member of this family. Based on the substrate-binding orientation and substrate specificity, PcRGLX is an exo-type PL that cleaves rhamnogalacturonan from the reducing end. Analysis of PcRGLX-complex structures with reaction products indicate that the active site possesses an L-shaped cleft that can accommodate galactosyl side chains, suggesting side-chain-bypassing activity in PcRGLX. Furthermore, we determined the residues critical for catalysis by analyzing the enzyme activities of inactive variants.

Structural and functional analysis of tomato ß-galactosidase 4: insight into the substrate specificity of the fruit softening-related enzyme.

Plant ß-galactosidases hydrolyze cell wall ß-(1,4)-galactans to play important roles in cell wall expansion and degradation, and turnover of signaling molecules, during ripening. Tomato ß-galactosidase 4 (TBG4) is an enzyme responsible for fruit softening through the degradation of ß-(1,4)-galactan in the pericarp cell wall. TBG4 is the only enzyme among TBGs 1-7 that belongs to the ß-galactosidase/exo-ß-(1,4)-galactanase subfamily. The enzyme can hydrolyze a wide range of plant-derived (1,4)- or 4-linked polysaccharides, and shows a strong ability to attack ß-(1,4)-galactan. To gain structural insight into its substrate specificity, we determined crystal structures of TBG4 and its complex with ß-d-galactose. TBG4 comprises a catalytic TIM barrel domain followed by three ß-sandwich domains. Three aromatic residues in the catalytic site that are thought to be important for substrate specificity are conserved in GH35 ß-galactosidases derived from bacteria, fungi and animals; however, the crystal structures of TBG4 revealed that the enzyme has a valine residue (V548) replacing one of the conserved aromatic residues. The V548W mutant of TBG4 showed a roughly sixfold increase in activity towards ß-(1,6)-galactobiose, and ~0.6-fold activity towards ß-(1,4)-galactobiose, compared with wild-type TBG4. Amino acid residues corresponding to V548 of TBG4 thus appear to determine the substrate specificities of plant ß-galactosidases towards ß-1,4 and ß-1,6 linkages.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of tomato ß-galactosidase 4.

Plant ß-galactosidases play important roles in carbohydrate-reserve mobilization, cell-wall expansion and degradation, and turnover of signalling molecules during ripening. Tomato ß-galactosidase 4 (TBG4) not only has ß-galactosidase activity but also has exo-ß-(1,4)-galactanase activity, and prefers ß-(1,4)-galactans longer than pentamers as its substrates; most other ß-galactosidases only have the former activity. Recombinant TBG4 protein expressed in the yeast Pichia pastoris was crystallized by the sitting-drop vapour-diffusion method using PEG 10,000 as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-parameters a = 92.82, b = 96.30, c = 159.26 Å, and diffracted to 1.65 Å resolution. Calculation of the Matthews coefficient suggested the presence of two monomers per asymmetric unit (VM = 2.2 Å(3) Da(-1)), with a solvent content of 45%.

The crystal structure of isoniazid-bound KatG catalase-peroxidase from Synechococcus elongatus PCC7942.

Isoniazid (INH) is one of the most effective antibiotics against tuberculosis. INH is a prodrug that is activated by KatG. Although extensive studies have been performed in order to understand the mechanism of KatG, even the binding site of INH in KatG remains controversial. In this study, we determined the crystal structure of KatG from Synechococcus elongatus PCC7942 (SeKatG) in a complex with INH at 2.12-Šresolution. Three INH molecules were bound to the molecular surface. One INH molecule was bound at the entrance to the ε-edge side of heme (designated site 1), another was bound at the entrance to the γ-edge side of heme (site 2), and another was bound to the loop structures in front of the heme propionate side chain (site 3). All of the interactions between KatG and the bound INH seemed to be weak, being mediated mainly by van der Waals contacts. Structural comparisons revealed that the identity and configuration of the residues in site 1 were very similar among SeKatG, Burkholderia pseudomallei KatG, and Mycobacterium tuberculosis KatG. In contrast, sites 2 and 3 were structurally diverse among the three proteins. Thus, site 1 is probably the common KatG INH-binding site. A static enzymatic analysis and thermal shift assay suggested that the INH-activating reaction does not proceed in site 1, but rather that this site may function as an initial trapping site for the INH molecule. Database: The atomic coordinates and structure factors have been deposited in the Protein Data Bank under the accession number 3WXO.

Crystal structure of the catalase-peroxidase KatG W78F mutant from Synechococcus elongatus PCC7942 in complex with the antitubercular pro-drug isoniazid.

Isoniazid (INH) is a pro-drug that has been extensively used to treat tuberculosis. INH is activated by the heme enzyme catalase-peroxidase (KatG), but the mechanism of the activation is poorly understood, in part because the INH binding site has not been clearly established. Here, we observed that a single-residue mutation of KatG from Synechococcus elongatus PCC7942 (SeKatG), W78F, enhances INH activation. The crystal structure of INH-bound KatG-W78F revealed that INH binds to the heme pocket. The results of a thermal-shift assay implied that the flexibility of the SeKatG molecule is increased by the W78F mutation, allowing the INH molecule to easily invade the heme pocket through the access channel on the γ-edge side of the heme.

Enzymatic activity and substrate specificity of the recombinant tomato ß-galactosidase 1.

The open reading frame of tomato ß-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on ß-(1,3)- and ß-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl ß-(1,6)-galactohexaoside, but not from labeled ß-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

Crystallization and preliminary X-ray diffraction studies of La1 from Liocheles australasiae.

A novel scorpion venom peptide, La1 from Liocheles australasiae, with a molecular weight of 7.8 kDa, is presumed to possess a single von Willebrand factor type C (VWC) domain, a common protein module, based on the position of eight Cys residues in its sequence. The biological function of La1 is still unknown. Deciphering its three-dimensional structure will be helpful in understanding its biological function. La1 was crystallized by the sitting-drop vapour-diffusion method using magnesium sulfate as a precipitant. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=63.0, b=30.2, c=32.3 Å, ß=108.5°, and diffracted to 1.9 Šresolution. The calculated VM based on one molecule per asymmetric unit was 1.87 Å3 Da(-1). The solvent content was 34.1%.

The 2.2†Šresolution structure of the catalase-peroxidase KatG from Synechococcus elongatus PCC7942.

The crystal structure of catalase-peroxidase from Synechococcus elongatus PCC7942 (SeKatG) was solved by molecular replacement and refined to an Rwork of 16.8% and an Rfree of 20.6% at 2.2 Šresolution. The asymmetric unit consisted of only one subunit of the catalase-peroxidase molecule, including a protoporphyrin IX haem moiety and two sodium ions. A typical KatG covalent adduct was formed, Met248-Tyr222-Trp94, which is a key structural element for catalase activity. The crystallographic equivalent subunit was created by a twofold symmetry operation to form the functional dimer. The overall structure of the dimer was quite similar to other KatGs. One sodium ion was located close to the proximal Trp314. The location and configuration of the proximal cation site were very similar to those of typical peroxidases such as ascorbate peroxidase. These features may provide a structural basis for the behaviour of the radical localization/delocalization during the course of the enzymatic reaction.

Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

Seven cysteine-deficient mutants depict the interplay between thermal and chemical stabilities of individual cysteine residues in mitogen-activated protein kinase c-Jun N-terminal kinase 1.

Intracellular proteins can have free cysteines that may contribute to their structure, function, and stability; however, free cysteines can lead to chemical instabilities in solution because of oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase 1 (JNK1), possesses seven free cysteines and is an important drug target for autoimmune diseases, cancers, and apoptosis-related diseases. To characterize the role of cysteine residues in the structure, function, and stability of JNK1, we prepared and evaluated wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments showed that the chemical stability of JNK1 increased as the number of cysteines decreased. The contribution of each cysteine residue to biological function and thermal stability was highly susceptible to the environment surrounding the particular cysteine mutation. The mutations of solvent-exposed cysteine to serine did not influence biological function and increased the thermal stability. The mutation of the accessible cysteine involved in the hydrophobic pocket did not affect biological function, although a moderate thermal destabilization was observed. Cysteines in the loosely assembled hydrophobic environment moderately contributed to thermal stability, and the mutations of these cysteines had a negligible effect on enzyme activity. The other cysteines are involved in the tightly filled hydrophobic core, and mutation of these residues was found to correlate with thermal stability and enzyme activity. These findings about the role of cysteine residues should allow us to obtain a stable JNK1 and thus promote the discovery of potent JNK1 inhibitors.

Crystal and solution structures disclose a putative transient state of mitogen-activated protein kinase kinase 4.

Mitogen-activated protein kinase kinase 4 (MAP2K4) plays a crucial role in the stress-activated signal cascade and is enzymatically regulated by ligand or substrate binding, and/or post-translational modification. Crystal structures combined with small-angle X-ray scattering experiments revealed that the apo form of non-phosphorylated MAP2K4 (npMAP2K4) exists in a transient state which has a longer conformation compared with the typical kinase folding. Upon ATP-binding, the transient conformation adopted the configuration of typical kinase folding. In the absence of ATP-binding, the transient state of apo npMAP2K4 may shift to a state of aggregation via non-particular hydrophobic interactions as a result of the exposed hydrophobic residues.

Crystal structure of non-phosphorylated MAP2K6 in a putative auto-inhibition state.

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in the p38 MAP kinase signal cascade that regulates various stress-induced responses and is associated with pathological conditions. The crystal structure of human non-phosphorylated MAP2K6 (npMAP2K6) complexed with an ATP analogue was determined at 2.6 Å resolution and represents an auto-inhibition state of MAP2K6. Three characteristics of short α-helices configured in the activation loop region, termed activation helices (AH1, AH2 and AH3), are important in controlling the auto-inhibition mechanism. AH1 displaces the αC-helix, a component essential for forming the active configuration, away from the active site. AH1 and AH2 were found to enclose the γ-phosphate, the leaving group of ATP. A comparison with the related enzymes, MAP2K1 and MAP2K4 reveals that MAP2K6 has the unique auto-inhibition mechanism mediated by the three activation helices.

Biochemical characterization and gene expression of two endo-arabinanases from Penicillium chrysogenum 31B.

We previously described five arabinanolytic enzymes secreted by Penicillium chrysogenum 31B into the culture medium. Here, we describe a sixth such enzyme, termed AbnS1. Analysis of the reaction products of debranched arabinan revealed that AbnS1 cleaved the substrate in an endo manner. The optimum temperature of AbnS1 was 60°C, which was much higher than that of a cold-adapted endo-arabinanase (Abnc) produced by this strain. The abns1 cDNA gene encoding AbnS1 was isolated by in vitro cloning. The deduced amino acid sequence of AbnS1 had 70% identity with that of Abnc. Pfam analysis revealed a Glyco_hydro_43 domain at positions 28 to 318 of AbnS1. Semi-quantitative reverse transcription-polymerase chain reaction analysis indicated that the abns1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was only slightly induced with arabinose and arabinan. In contrast, expression of the abnc gene encoding Abnc was strongly induced by arabinose, arabinitol, and arabinan. Using debranched arabinan as substrate, recombinant AbnS1 (rAbnS1) accumulated arabinobiose and arabinotriose as the major products. Recombinant Abnc (rAbnc) released mainly arabinotriose and lesser amounts of arabinose and arabinobiose than did rAbnS1. Branched arabinan was completely degraded to arabinose by the action of rAbnS1 or rAbnc in combination with α-L: -arabinofuranosidase.

A detailed thermodynamic profile of cyclopentyl and isopropyl derivatives binding to CK2 kinase.

The detailed understanding of the molecular features of a ligand binding to a target protein, facilitates the successful design of potent and selective inhibitors. We present a case study of ATP-competitive kinase inhibitors that include a pyradine moiety. These compounds have similar chemical structure, except for distinct terminal hydrophobic cyclopentyl or isopropyl groups, and block kinase activity of casein kinase 2 subunit α (CK2α), which is a target for several diseases, such as cancer and glomerulonephritis. Although these compounds display similar inhibitory potency against CK2α, the crystal structures reveal that the cyclopentyl derivative gains more favorable interactions compared with the isopropyl derivative, because of the additional ethylene moiety. The structural observations and biological data are consistent with the thermodynamic profiles of these inhibitors in binding to CK2α, revealing that the enthalpic advantage of the cyclopentyl derivative is accompanied with a lower entropic loss. Computational analyses indicated that the relative enthalpic gain of the cyclopentyl derivative arises from an enhancement of a wide range of van der Waals interactions from the whole complex. Conversely, the relative entropy loss of the cyclopentyl derivative arises from a decrease in the molecular fluctuation and higher conformational restriction in the active site of CK2α. These structural insights, in combination with thermodynamic and computational observations, should be helpful in developing potent and selective CK2α inhibitors.

High-resolution structure of exo-arabinanase from Penicillium chrysogenum.

Arabinanase Abnx from Penicillium chrysogenum 31B, which belongs to the GH93 family, releases arabinobiose from the nonreducing terminus of α-1,5-L-arabinan, which is distributed in the primary cell walls of higher plants. Crystal structures of Abnx and of its complex with arabinobiose were determined at the high resolutions of 1.14 Što an R(work) of 10.7% (R(free) = 12.8%) and 1.04 Što an R(work) of 10.4% (R(free) = 12.5%). Abnx has a six-bladed ß-propeller fold with a typical ring-closure mode called `Velcro', in which the last four-stranded ß-sheet is completed by the incorporation of a strand from the N-terminus. Catalytic residues which act as a nucleophile and an acid/base were proposed from the structures and confirmed by site-directed mutagenesis. The substrate-binding groove is enclosed at one end by two residues, Glu64 and Tyr66, which contribute to the recognition of the nonreducing chain end of the polysaccharide. A comparison with the related enzyme Arb93A which has a quite similar overall structure suggested that Abnx has different mechanisms to funnel substrates to the active site and/or to stabilize the transition state.

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms.

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by ß-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded ß-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to ß-strand 7 and measured T(m) values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state.

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.

A silent mutation made possible efficient production of active human Frk tyrosine kinase in Escherichia coli.

Fyn-related kinase (Frk) was first identified using human breast cancer cells. It shares 51% identity with c-Src. Like all members of the Src family, Frk is thought to cause several cancers via dysregulations in signal transduction from cell-surface receptors. The excess activity of Frk on beta-cells has a crucial role in type-I diabetes. A silent mutation at Ile229 conferred a bacterial expression system on the kinase domains of Frk, which allowed for the quick expression and purification of one unphosphorylated and two mono-phosphorylated kinase domains. The C-terminal catalytic segment of the human Frk kinase conjugating hexahistidine purification tag (His-tag) was expressed in Escherichia coli. After first-step purification utilizing the His-tag, an anion-exchange chromatogram yielded three major peaks that had distinguishable phosphorylation characteristics as judged by Western blot analysis and measurement of kinase activity. This result of active protein production should promote drug discovery studies, including highthrough-put screening and structure-based drug design.