Comprehensive Approach of 19F Nuclear Magnetic Resonance, Enzymatic, and In Silico Methods for Site-Specific Hit Selection and Validation of Fragment Molecules that Inhibit Methionine γ-Lyase Activity.

Fragment-based screening using 19F NMR (19F-FS) is an efficient method for exploring seed and lead compounds for drug discovery. Here, we demonstrate the utility and merits of using 19F-FS for methionine γ-lyase-binding fragments, together with a 19F NMR-based competition and mutation assay, as well as enzymatic and in silico methods. 19F NMR-based assays provided useful information on binding between 19F-FS hit fragments and target proteins. Although the 19F-FS and enzymatic assay were weakly correlated, they show that the 19F-FS hit fragments contained compounds with inhibitory activity. Furthermore, we found that in silico calculations partially account for the differences in activity levels between the 19F-FS hits as per NMR analysis. A comprehensive approach combining the 19F-FS and other methods not only identified fragment hits but also distinguished structural differences in chemical groups with diverse activity levels.

Identification and Analysis of a Novel NR0B1 Mutation in Late-Onset Adrenal Hypoplasia Congenita and Hypogonadism.

OBJECTIVE: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency and hypogonadotropic hypogonadism (HHG) caused by mutations of the NR0B1/DAX1 gene. We aimed to search for the presence of any NR0B1/DAX1 gene mutations in a referred patient and to further characterize the phenotypes of the identified mutation. CASE PRESENTATION: Herein, we report a Japanese patient with a novel missense mutation of the NR0B1/DAX1 gene resulting in adult-onset AHC and HHG. The patient was referred with diffuse skin pigmentation at 28 years of age, presented partial adrenal insufficiency and had undiagnosed incomplete HHG. Urological examination revealed severe oligospermia and testicular microlithiasis. RESULTS: The NR0B1/DAX1 gene mutation was identified by exome sequencing as a novel missense mutation (c.884A>T, p.Leu295His). We conducted in silico modeling of this mutant NR0B1/DAX1 protein (p.Leu295His) which affected the conserved hydrophobic core of the putative ligand-binding domain (LBD). In addition, functional analysis revealed that this mutant showed a decreased ability as a transcriptional repressor to suppress target genes, such as STAR and LHB. Furthermore, this mutant showed functionally impaired repression of steroidogenesis in human adrenocortical H295R cells. CONCLUSIONS: We identified a novel missense mutation of the NR0B1/DAX1 gene in a patient suffering from late-onset AHC and HHG, who presented with oligospermia and testicular microlithiasis. This mutant NR0B1/DAX1 protein was found to have reduced repressor activity, according to in vitro studies, clinically consistent with the patient's phenotypic features.

Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis.

Acetyl phosphate (AcP) is generally produced from acetyl coenzyme A by phosphotransacetylase (Pta), and subsequent reaction with ADP, catalyzed by acetate kinase (Ack), produces ATP. The mechanism of ATP production in Porphyromonas gingivalis is poorly understood. The aim of this study was to explore the molecular basis of the Pta-Ack pathway in this microorganism. Pta and Ack from P. gingivalis ATCC 33277 were enzymatically and structurally characterized. Structural and mutational analyses suggest that Pta is a dimer with two substrate-binding sites in each subunit. Ack is also dimeric, with a catalytic cleft in each subunit, and structural analysis indicates a dramatic domain motion that opens and closes the cleft during catalysis. ATP formation by Ack proceeds via a sequential mechanism. Reverse transcription-PCR analysis demonstrated that the pta (PGN_1179) and ack (PGN_1178) genes, tandemly located in the genome, are cotranscribed as an operon. Inactivation of pta or ack in P. gingivalis by homologous recombination was successful only when the inactivated gene was expressed in trans. Therefore, both pta and ack genes are essential for this microorganism. Insights into the Pta-Ack pathway reported herein would be helpful to understand the energy acquisition in P. gingivalis.

Physical and thermodynamic characterization of the rice gibberellin receptor/gibberellin/DELLA protein complex.

Gibberellins (GAs) are phytohormones that regulate various developmental processes in plants. The initial GA signalling events involve the binding of a GA to the soluble GA receptor protein GID1, followed by the binding of the complex to the negative transcriptional regulator of GA signaling, the DELLA protein. Although X-ray structures for certain Arabidopsis GID1/GA/DELLA protein complexes have previously been determined, examination of these complexes did not fully clarify how a DELLA protein recognizes and binds to a GID1/GA complex. Herein, we present a study aimed at physically defining, via a combination of gel chromatography, isothermal titration calorimetry (ITC), small-angle X-ray scattering experiments (SAXS), NMR spectroscopy and mutagenesis, how the rice DELLA protein (SLR1) binds to the rice GID1/GA complex. We have identified the shortest SLR1 sequence (M28-A112) that binds the rice GID/GA complex tightly. The binding constant for the ternary complex that includes SLR1(M28-A112) is 2.9 × 107 M-1; the binding is enthalpically driven and does not depend on the chemical nature of the bound GA. Furthermore, the results of SAXS, ITC, and gel filtration experiments indicate that when free in solution, SLR1(M28-A112) is a natively unfolded protein. The NMR experiments expand this observation to show that the unfolded mutant also contains a small amount of marginally stable secondary structure. Conversely, the protein has a highly ordered structure when bound one-to-one to GID1/GA.

Structural insights into the catalytic mechanism of cysteine (hydroxyl) lyase from the hydrogen sulfide-producing oral pathogen, Fusobacterium nucleatum.

Hydrogen sulfide (H2S) plays important roles in the pathogenesis of periodontitis. Oral pathogens typically produce H2S from l-cysteine in addition to pyruvate and [Formula: see text] However, fn1055 from Fusobacterium nucleatum subsp. nucleatum ATCC 25586 encodes a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the production of H2S and l-serine from l-cysteine and H2O, an unusual cysteine (hydroxyl) lyase reaction (ß-replacement reaction). To reveal the reaction mechanism, the crystal structure of substrate-free Fn1055 was determined. Based on this structure, a model of the l-cysteine-PLP Schiff base suggested that the thiol group forms hydrogen bonds with Asp232 and Ser74, and the substrate α-carboxylate interacts with Thr73 and Gln147 Asp232 is a unique residue to Fn1055 and its substitution to asparagine (D232N) resulted in almost complete loss of ß-replacement activity. The D232N structure obtained in the presence of l-cysteine contained the α-aminoacrylate-PLP Schiff base in the active site, indicating that Asp232 is essential for the addition of water to the α-aminoacrylate to produce the l-serine-PLP Schiff base. Rapid-scan stopped-flow kinetic analyses showed an accumulation of the α-aminoacrylate intermediate during the reaction cycle, suggesting that water addition mediated by Asp232 is the rate-limiting step. In contrast, mutants containing substitutions of other active-site residues (Ser74, Thr73, and Gln147) exhibited reduced ß-replacement activity by more than 100-fold. Finally, based on the structural and biochemical analyses, we propose a mechanism of the cysteine (hydroxyl) lyase reaction by Fn1055. The present study leads to elucidation of the H2S-producing mechanism in F. nucleatum.

Acyl-CoA reductase PGN_0723 utilizes succinyl-CoA to generate succinate semialdehyde in a butyrate-producing pathway of Porphyromonas gingivalis.

The molecular basis of butyrate production in Porphyromonas gingivalis has not been fully elucidated, even though butyrate, a short chain fatty acid (SCFA), can exert both beneficial and harmful effects on a mammalian host. A database search showed that the amino acid sequence of PGN_0723 protein was 50.6% identical with CoA-dependent succinate semialdehyde dehydrogenase (SSADH) in Clostridium kluyveri. By contrast, the protein has limited identity (19.1%) with CoA-independent SSADH in Escherichia coli. Compared with the wild type, growth speed, and final turbidity were lower in the PGN_0723 deletion strain that was constructed by replacing the PGN_0723 gene with an erythromycin resistance cassette. Gas chromatography mass spectrometry revealed the supernatant concentrations of the SCFAs butyrate, isobutyrate, and isovalerate, but not propionate, in the PGN_0723 deletion strain were also lower than those in the wild type. The wild-type phenotype was restored in a complemented strain. We cloned the PGN_0723 gene, purified the recombinant protein, and computationally constructed its three-dimensional model. A colorimetric assay and liquid chromatography-tandem mass spectrometry analysis demonstrated that the recombinant PGN_0723 produces succinate semialdehyde, which is an intermediate in the P. gingivalis butyrate synthesis pathway, not from succinate but from succinyl-CoA in the presence of NAD(P)H via a ping-pong bi-bi mechanism. Asn110Ala and Cys239Ala mutations resulted in a significant loss of the CoA-dependent PGN_0723 enzymatic activity. The study provides new insights into butyrate production, which constitutes a virulence factor in P. gingivalis.

Structural basis for the recognition-evasion arms race between Tomato mosaic virus and the resistance gene Tm-1.

The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differences between ToMV-Hel in its free form and in complex with Tm-1 suggest that Tm-1 affects nucleoside triphosphatase activity of ToMV-Hel, and this effect was confirmed experimentally. Molecular dynamics simulations of complexes formed by Tm-1 with ToMV-Hel variants showed how the amino acid changes in ToMV-Hel impair the interaction with Tm-1 to overcome the resistance. With these findings, together with the biochemical properties of the interactions between ToMV-Hel and Tm-1 variants and effects of the mutations in the polymorphic residues of Tm-1, an atomic view of a step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerges.

Crystallization and preliminary X-ray crystallographic analysis of the inhibitory domain of the tomato mosaic virus resistance protein Tm-1.

Tm-1, an inhibitor protein of Tomato mosaic virus RNA replication, contains two conserved domains: an uncharacterized domain at its N-terminus and a TIM-barrel-like domain at its C-terminus. The N-terminal domain of Tm-1 has an inhibitory activity and its three-dimensional structure has not been determined. Here, the crystallization and preliminary X-ray diffraction of the N-terminal domain of Tm-1 are reported. A three-wavelength MAD data set was collected from a selenomethionine-labelled crystal and processed to 2.7 Å resolution. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 77.97, b = 105.28, c = 110.62 Å, α = 94.6, ß = 109.3, γ = 108.0°.

Purification, crystallization and preliminary X-ray analysis of two hydrogen sulfide-producing enzymes from Fusobacterium nucleatum.

Hydrogen sulfide produced by oral bacteria is responsible for oral malodour. Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from Fusobacterium nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. X-ray diffraction data were collected from the crystals using a synchrotron-radiation source. The Fn1220 crystal belonged to tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 (unit-cell parameters a=b=116.8, c=99.2 Å) and the Cdl crystal belonged to monoclinic space group P2(1) (unit-cell parameters a=84.9, b=70.9, c=87.6 Å, ß=90.3°).

Structural insights into catalysis by ßC-S lyase from Streptococcus anginosus.

Hydrogen sulfide (H(2)S) is a causative agent of oral malodor and may play an important role in the pathogenicity of oral bacteria such as Streptococcus anginosus. In this microorganism, H(2)S production is associated with ßC-S lyase (Lcd) encoded by lcd gene, which is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the α,ß-elimination of sulfur-containing amino acids. When Lcd acts on L-cysteine, H(2)S is produced along with pyruvate and ammonia. To understand the H(2)S-producing mechanism of Lcd in detail, we determined the crystal structures of substrate-free Lcd (internal aldimine form) and two reaction intermediate complexes (external aldimine and α-aminoacrylate forms). The formation of intermediates induced little changes in the overall structure of the enzyme and in the active site residues, with the exception of Lys234, a PLP-binding residue. Structural and mutational analyses highlighted the importance of the active site residues Tyr60, Tyr119, and Arg365. In particular, Tyr119 forms a hydrogen bond with the side chain oxygen atom of L-serine, a substrate analog, in the external aldimine form suggesting its role in the recognition of the sulfur atom of the true substrate (L-cysteine). Tyr119 also plays a role in fixing the PLP cofactor at the proper position during catalysis through binding with its side chain. Finally, we partly modified the catalytic mechanism known for cystalysin, a ßC-S lyase from Treponema denticola, and proposed an improved mechanism, which seems to be common to the ßC-S lyases from oral bacteria.

Identification and enzymic analysis of a novel protein associated with production of hydrogen sulfide and L-serine from L-cysteine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

A third enzyme that produces hydrogen sulfide from L-cysteine was identified in Fusobacterium nucleatum subsp. nucleatum. The fn1055 gene was cloned from a cosmid library constructed with genomic DNA of F. nucleatum ATCC 25586. Despite the database annotation that the product of fn1055 is a cysteine synthase, reverse-phase HPLC revealed that no L-cysteine was produced in vitro by the purified Fn1055 protein; however, the enzyme did produce L-serine. In addition, a cysteine auxotroph, Escherichia coli NK3, transformed with a plasmid containing the fn1055 gene did not grow without cysteine, which further suggests that Fn1055 does not function as a cysteine synthase. The Michaelis-Menten kinetics (K(m) =0.09 ± 0.001 mM and k(cat) =5.43 ± 0.64 s(-1)) of the purified enzyme showed that the capacity of Fn1055 to produce hydrogen sulfide was between that of two other enzymes, Fn0625 and Fn1220. Incubation of Fn1055 with L-cysteine resulted in the production of hydrogen sulfide, but not of pyruvate, ammonia or lanthionine, which are all byproducts produced in addition to hydrogen sulfide when Fn0625 or Fn1220 is incubated with L-cysteine. Instead, Fn1055 produced L-serine in its reaction with L-cysteine. Fn1055 produces hydrogen sulfide from l-cysteine by a mechanism that is different from that of Fn0625 or Fn1220.

Thermodynamic characterization of OsGID1-gibberellin binding using calorimetry and docking simulations.

Gibberellins (GAs) are phytohormones regulating various developmental processes in plants. In rice, the initial GA-signaling events involve the binding of a GA to the soluble GA receptor protein, GID1. Although X-ray structures for certain GID1/GA complexes have recently been determined, an examination of the complexes does not fully clarify how GID1s discriminate among different GAs. Herein, we present a study aimed at defining the types of forces important to binding via a combination of isothermal titration calorimetry (ITC) and computational docking studies that employed rice GID1 (OsGID1), OsGID1 mutants, which were designed to have a decreased possible number of hydrogen bonds with bound GA, and GA variants. We find that, in general, GA binding is enthalpically driven and that a hydrogen bond between the phenolic hydroxyl of OsGID1 Tyr134 and the C-3 hydroxyl of a GA is a defining structural element. A hydrogen-bond network that involves the C-6 carboxyl of a GA that directly hydrogen bonds the hydroxyl of Ser198 and indirectly, via a two-water-molecule network, the phenolic hydroxyl of Tyr329 and the NH of the amide side-chain of Asn255 is also important for GA binding. The binding of OsGID1 by GA(1) is the most enthalpically driven association found for the biologically active GAs evaluated in this study. This observation might be a consequence of a hydrogen bond formed between the hydroxyl at the C-13 position of GA(1) and the main chain carbonyl of OsGID1 Phe245. Our results demonstrate that by combining ITC experiments and computational methods much can be learned about the thermodynamics of ligand/protein binding.

Structure of full-length class I chitinase from rice revealed by X-ray crystallography and small-angle X-ray scattering.

The rice class I chitinase OsChia1b, also referred to as RCC2 or Cht-2, is composed of an N-terminal chitin-binding domain (ChBD) and a C-terminal catalytic domain (CatD), which are connected by a proline- and threonine-rich linker peptide. Because of the ability to inhibit fungal growth, the OsChia1b gene has been used to produce transgenic plants with enhanced disease resistance. As an initial step toward elucidating the mechanism of hydrolytic action and antifungal activity, the full-length structure of OsChia1b was analyzed by X-ray crystallography and small-angle X-ray scattering (SAXS). We determined the crystal structure of full-length OsChia1b at 2.00-A resolution, but there are two possibilities for a biological molecule with and without interdomain contacts. The SAXS data showed an extended structure of OsChia1b in solution compared to that in the crystal form. This extension could be caused by the conformational flexibility of the linker. A docking simulation of ChBD with tri-N-acetylchitotriose exhibited a similar binding mode to the one observed in the crystal structure of a two-domain plant lectin complexed with a chitooligosaccharide. A hypothetical model based on the binding mode suggested that ChBD is unsuitable for binding to crystalline alpha-chitin, which is a major component of fungal cell walls because of its collisions with the chitin chains on the flat surface of alpha-chitin. This model also indicates the difference in the binding specificity of plant and bacterial ChBDs of GH19 chitinases, which contribute to antifungal activity.

Production of hydrogen sulfide by two enzymes associated with biosynthesis of homocysteine and lanthionine in Fusobacterium nucleatum subsp. nucleatum ATCC 25586.

Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H(2)S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H(2)S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H(2)S from L-cysteine (approximately 30%) than Fn1220. The Fn0625 protein degraded a variety of substrates containing betaC-S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from L-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of L-cysteine with Fn1220 produced H(2)S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except L-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H(2)S in distinct manners: Fn0625 carries out beta-elimination of L-cysteine to produce H(2)S, pyruvate and ammonia, whereas Fn1220 catalyses the beta-replacement of L-cysteine to produce H(2)S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.

Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release.

Functional investigation of the proposed dehydratase domain of ATX, a 6-methylsalicylic acid synthase from Aspergillus terreus, revealed that the domain is not involved in dehydration of the beta-hydroxytriketide intermediate tethered on the acyl carrier protein but catalyzes thioester hydrolysis to release the product from the acyl carrier protein. Thus, we renamed this domain the thioester hydrolase (TH) domain. The intermediate bound to the TH domain of mutant H972A formed in the presence of NADPH was released as 6-methylsalicylic acid by both the intact ATX and by THID (a 541-amino acid region containing TH domain and its downstream) protein, in trans. Furthermore, THID showed a catalytic activity to hydrolyze a model substrate, 6-methylsalicylic acid-N-acetylcysteamine. The TH domain is the first example of a product-releasing domain that is located in the middle of a multidomain iterative type I polyketide synthase. Moreover, it is functionally different from serine protease-type thioesterase domains of iterative type I polyketide synthases.

Crystallization and preliminary X-ray analysis of betaC-S lyases from two oral Streptococci.

Hydrogen sulfide, which causes oral malodour, is generally produced from L-cysteine by the action of betaC-S lyase from oral bacteria. The betaC-S lyases from two oral bacteria, Streptococcus anginosus and S. gordonii, have been cloned, overproduced, purified and crystallized. X-ray diffraction data were collected from the two types of crystals using synchrotron radiation. The crystal of S. anginosus betaC-S lyase belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.0, b = 111.1, c = 216.4 A, and the crystal of S. gordonii betaC-S lyase belonged to the same space group, with unit-cell parameters a = 58.0, b = 73.9. c = 187.6 A. The structures of the betaC-S lyases were solved by molecular-replacement techniques.

Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen.

A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

SaxsMDView: a three-dimensional graphics program for displaying force vectors.

A new three-dimensional graphics program, SaxsMDView, is described. The program performs a three-dimensional graphical representation for protein molecules along with the force vector (or vector potential) applying to each atom. The displayed object can be rotated and translated in arbitrary directions by interactive mouse manipulation. While SaxsMDView was originally intended to visualize the result of SAXS_MD, a previously developed program based on the restrained molecular dynamics with small-angle X-ray scattering constraints, it can also be useful for graphical representation of other objects such as coarse-grained molecular models reconstructed by ab initio modelling or solvent site-dipole field vectors induced around the protein molecule. Some examples of the application of the program including the graphical analyses of the results with SAXS_MD are also presented.

Crystallization and preliminary x-ray crystallographic analysis of galectin LEC-1 from Caenorhabditis elegans.

Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 A, alpha = 108.73, beta= 91.39, and gamma = 98.45 degrees and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 A, and beta= 113.30 degrees and one protein molecule per asymmetric unit.

Purification and characterization of a rice class I chitinase, OsChia1b, produced in Esherichia coli.

To determine the properties and structure of OsChia1b, a family 19 chitinase from Oryza sativa L. cv. Nipponbare (japonica ssp.), recombinant OsChia1b was produced in Esherichia coli cells and purified to homogeneity by chitin affinity column chromatography. OsChia1b was highly active against soluble chitinous substrate, but not against crystalline chitin, and clearly inhibited hyphal extension of Trichoderma reesei.