RESUMEN
The crystal structure of l-lactate oxidase in complex with l-lactate was solved at a 1.33 Å resolution. The electron density of the bound l-lactate was clearly shown and comparisons of the free form and substrate bound complexes demonstrated that l-lactate was bound to the FMN and an additional active site within the enzyme complex. l-lactate interacted with the related side chains, which play an important role in enzymatic catalysis and especially the coupled movement of H265 and D174, which may be essential to activity. These observations not only reveal the enzymatic mechanism for l-lactate binding but also demonstrate the dynamic motion of these enzyme structures in response to substrate binding and enzymatic reaction progression.
Asunto(s)
Aerococcus/metabolismo , Proteínas Bacterianas/metabolismo , Ácido Láctico/metabolismo , Oxigenasas de Función Mixta/metabolismo , Aerococcus/química , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Ácido Láctico/química , Oxigenasas de Función Mixta/química , Modelos Moleculares , Especificidad por SustratoRESUMEN
Band 3 mediates the Cl- and HCO3- exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study. In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station "KIBO" under microgravity environments.
Asunto(s)
Proteína 1 de Intercambio de Anión de Eritrocito/química , Proteína 1 de Intercambio de Anión de Eritrocito/aislamiento & purificación , Cristalización/métodos , Nave Espacial , Ingravidez , Cristalografía por Rayos X/métodos , Membrana Eritrocítica/química , HumanosRESUMEN
Hematopoietic prostaglandin D synthase (H-PGDS) is one of the two enzymes that catalyze prostaglandin D2 synthesis and a potential therapeutic target of allergic and inflammatory responses. To reveal key molecular interactions between a high-affinity ligand and H-PGDS, we designed and synthesized a potent new inhibitor (KD: 0.14â¯nM), determined the crystal structure in complex with human H-PGDS, and quantitatively analyzed the ligand-protein interactions by the fragment molecular orbital calculation method. In the cavity, 10 water molecules were identified, and the interaction energy calculation indicated their stable binding to the surface amino acids in the cavity. Among them, 6 water molecules locating from the deep inner cavity to the peripheral part of the cavity contributed directly to the ligand binding by forming hydrogen bonding interactions. Arg12, Gly13, Gln36, Asp96, Trp104, Lys112 and an essential co-factor glutathione also had strong interactions with the ligand. A strong repulsive interaction between Leu199 and the ligand was canceled out by forming a hydrogen bonding network with the adjacent conserved water molecule. Our quantitative studies including crystal water molecules explained that compounds with an elongated backbone structure to fit from the deep inner cavity to the peripheral part of the cavity would have strong affinity to human H-PGDS.
Asunto(s)
Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/metabolismo , Agua/química , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/genética , Ligandos , Lipocalinas/antagonistas & inhibidores , Lipocalinas/genética , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Resonancia por Plasmón de Superficie , Termodinámica , Agua/metabolismoRESUMEN
Phycocyanobilin, a light-harvesting and photoreceptor pigment in higher plants, algae, and cyanobacteria, is synthesized from biliverdin IXα (BV) by phycocyanobilin:ferredoxin oxidoreductase (PcyA) via two steps of two-proton-coupled two-electron reduction. We determined the neutron structure of PcyA from cyanobacteria complexed with BV, revealing the exact location of the hydrogen atoms involved in catalysis. Notably, approximately half of the BV bound to PcyA was BVH(+), a state in which all four pyrrole nitrogen atoms were protonated. The protonation states of BV complemented the protonation of adjacent Asp105. The "axial" water molecule that interacts with the neutral pyrrole nitrogen of the A-ring was identified. His88 Nδ was protonated to form a hydrogen bond with the lactam O atom of the BV A-ring. His88 and His74 were linked by hydrogen bonds via H3O(+). These results imply that Asp105, His88, and the axial water molecule contribute to proton transfer during PcyA catalysis.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biliverdina/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Synechocystis/enzimología , Cristalografía , Cristalografía por Rayos X , Modelos Moleculares , Difracción de Neutrones , Protones , Synechocystis/química , Synechocystis/metabolismoRESUMEN
Recently, many technical improvements in macromolecular X-ray crystallography have increased the number of structures deposited in the Protein Data Bank and improved the resolution limit of protein structures. Almost all high-resolution structures have been determined using a synchrotron radiation source in conjunction with cryocooling techniques, which are required in order to minimize radiation damage. However, optimization of cryoprotectant conditions is a time-consuming and difficult step. To overcome this problem, the high-pressure cryocooling method was developed (Kim et al., 2005) and successfully applied to many protein-structure analyses. In this report, using the high-pressure cryocooling method, the X-ray crystal structure of bovine H-protein was determined at 0.86 Å resolution. Structural comparisons between high- and ambient-pressure cryocooled crystals at ultra-high resolution illustrate the versatility of this technique. This is the first ultra-high-resolution X-ray structure obtained using the high-pressure cryocooling method.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Animales , Bovinos , Frío , Modelos Moleculares , Conformación ProteicaRESUMEN
The Japan Aerospace Exploration Agency (JAXA) started a high-quality protein crystal growth project, now called JAXA PCG, on the International Space Station (ISS) in 2002. Using the counter-diffusion technique, 14 sessions of experiments have been performed as of 2012 with 580 proteins crystallized in total. Over the course of these experiments, a user-friendly interface framework for high accessibility has been constructed and crystallization techniques improved; devices to maximize the use of the microgravity environment have been designed, resulting in some high-resolution crystal growth. If crystallization conditions were carefully fixed in ground-based experiments, high-quality protein crystals grew in microgravity in many experiments on the ISS, especially when a highly homogeneous protein sample and a viscous crystallization solution were employed. In this article, the current status of JAXA PCG is discussed, and a rational approach to high-quality protein crystal growth in microgravity based on numerical analyses is explained.
Asunto(s)
Proteínas/química , Vuelo Espacial , Cristalización , JapónRESUMEN
It is said that the microgravity environment positively affects the quality of protein crystal growth. The formation of a protein depletion zone and an impurity depletion zone due to the suppression of convection flow were thought to be the major reasons. In microgravity, the incorporation of molecules into a crystal largely depends on diffusive transport, so the incorporated molecules will be allocated in an orderly manner and the impurity uptake will be suppressed, resulting in highly ordered crystals. Previously, these effects were numerically studied in a steady state using a simplified model and it was determined that the combination of the diffusion coefficient of the protein molecule (D) and the kinetic constant for the protein molecule (ß) could be used as an index of the extent of these depletion zones. In this report, numerical analysis of these depletion zones around a growing crystal in a non-steady (i.e. transient) state is introduced, suggesting that this model may be used for the quantitative analysis of these depletion zones in the microgravity environment.
Asunto(s)
Cristalización , Muramidasa/química , Modelos Teóricos , IngravidezRESUMEN
Neutron protein crystallography (NPC) is a powerful tool for determining the hydrogen position and water orientation in proteins, but a much larger protein crystal is needed for NPC than for X-ray crystallography, and thus crystal preparation is a bottleneck. To obtain large protein crystals, it is necessary to know the properties of the target protein in the crystallization solution. Here, a crystal preparation method of fungal cellulase PcCel45A is reported, guided by the phase diagram. Nucleation and precipitation conditions were determined by sitting-drop vapor diffusion. Saturation and unsaturation conditions were evaluated by monitoring crystal dissolution, and a crystallization phase diagram was obtained. To obtain a large crystal, crystallization solution was prepared on a sitting bridge (diameter = 5 mm). Initial crystallization conditions were 40 µl of crystallization solution (40 mg ml(-1) protein with 30.5% 3-methyl-1,5-pentanediol in 50 mM tris-HCl pH 8.0) with a 1,000 µl reservoir (61% 3-methyl-1,5,-pentanediol in 50 mM tris-HCl pH 8.0) at 293 K. After the first crystal appeared, the concentration of precipitant in the reservoir solution was reduced to 60% to prevent formation of further crystals. Finally, we obtained a crystal of 6 mm(3) volume (3 mm × 2 mm × 1 mm), which was suitable for neutron diffraction.
Asunto(s)
Celulasa/química , Glicósido Hidrolasas/química , Cristalografía por Rayos X , NeutronesRESUMEN
Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophile Geobacillus collagenovorans MO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6â Å at 100â K from a crystal obtained in the Japanese Experiment Module (JEM; also known as `Kibo') at the International Space Station (ISS). The crystal belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 87.64, c = 210.5â Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.
Asunto(s)
Geobacillus/enzimología , Péptido Hidrolasas/química , Cristalización , Cristalografía por Rayos XRESUMEN
The structure determination of the PX (phox homology) domain of the Saccharomyces cerevisiae Vps17p protein presented a challenging case for molecular replacement because it has noncrystallographic symmetry close to a crystallographic axis. The combination of diffraction-quality crystals grown under microgravity on the International Space Station and a highly accurate template structure predicted by AlphaFold2 provided the key to successful crystal structure determination. Although the structure of the Vps17p PX domain is seen in many PX domains, no basic residues are found around the canonical phosphatidylinositol phosphate (PtdIns-P) binding site, suggesting an inability to bind PtdIns-P molecules.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sitios de Unión , Cristalografía por Rayos X , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme's catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min - much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.
Asunto(s)
Automonitorización de la Glucosa Sanguínea , Glucosa 1-Deshidrogenasa , Electrones , GlucemiaRESUMEN
Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosthetic group. The X-ray structures of OYE from Trypanosoma cruzi (TcOYE) which produces prostaglandin (PG) F(2α) from PGH(2) have been determined in the presence or absence of menadione. The binding motif of menadione, known as one of the inhibitors for TcOYE, should accelerate the structure-based development of novel anti-chagasic drugs that inhibit PGF(2α) production specifically.
Asunto(s)
Hidroxiprostaglandina Deshidrogenasas/antagonistas & inhibidores , NADPH Deshidrogenasa/antagonistas & inhibidores , NADPH Deshidrogenasa/química , Trypanosoma cruzi/enzimología , Vitamina K 3/química , Sitios de Unión , Cristalografía por Rayos X , Mononucleótido de Flavina/química , Hidroxiprostaglandina Deshidrogenasas/metabolismo , Modelos Moleculares , Vitamina K 3/farmacologíaRESUMEN
Although polyethylene glycol (PEG) is the most widely used precipitant in protein crystallization, the concentration of co-existing salt in the solution has not been well discussed. To determine the optimum salt concentration range, several kinds of protein were crystallized in a 30% PEG 4000 solution at various NaCl concentrations with various pH levels. It was found that, if crystallization occurred, the lowest effective salt concentration depended on the pH of the protein solution and the pI of the protein molecule; that is, higher salt concentrations were required for crystal growth if the difference between pH and pI was increasing. The linear relationship between the charge density of the protein and the ionic strength of the crystallization solution was further verified. These results suggested that the lowest effective concentration of salt in a crystallization solution can be predicted before performing a crystallization experiment. Our results can be a tip for tuning crystallization conditions by the vapor-diffusion method.
Asunto(s)
Cristalización/métodos , Polietilenglicoles/química , Cloruro de Sodio/química , Isomerasas Aldosa-Cetosa/química , Difusión , Concentración de Iones de Hidrógeno , Muramidasa/química , Concentración Osmolar , Soluciones , alfa-Amilasas/químicaRESUMEN
Human hematopoietic prostaglandin synthase, one of the better therapeutic target enzymes for allergy and inflammation, was crystallized with 22 inhibitors and in three inhibitor-free conditions in microgravity. Most of the space-grown crystals showed better X-ray diffraction patterns than the terrestrially grown ones, indicating the advantage of a microgravity environment on protein crystallization, especially in the case of this protein.
Asunto(s)
Cristalización/métodos , Oxidorreductasas Intramoleculares/química , Lipocalinas/química , Ingravidez , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Lipocalinas/antagonistas & inhibidores , Piperidinas/química , Vuelo Espacial , Difracción de Rayos XRESUMEN
Recent technical improvements in macromolecular X-ray crystallography have significantly improved the resolution limit of protein structures. However, examples of high-resolution structure determination are still limited. In this study, the X-ray crystal structure of bovine H-protein, a component of the glycine cleavage system, was determined at 0.88 A resolution. This is the first ultrahigh-resolution structure of an H-protein. The data were collected using synchrotron radiation. Because of limitations of the hardware, especially the dynamic range of the CCD detector, three data sets (high-, medium- and low-resolution data sets) were measured in order to obtain a complete set of data. To improve the quality of the merged data, the reference data set was optimized for merging and the merged data were assessed by comparing merging statistics and R factors against the final model and the number of visualized H atoms. In addition, the advantages of merging three data sets were evaluated. The omission of low-resolution reflections had an adverse effect on visualization of H atoms in hydrogen-omit maps. Visualization of hydrogen electron density is a good indicator for assessing the quality of high-resolution X-ray diffraction data.
Asunto(s)
Proteína H del Complejo de la Glicina Descarboxilasa/química , Animales , Bovinos , Cristalografía por Rayos X , Modelos Moleculares , Estructura Terciaria de Proteína , ProtonesRESUMEN
Human haematopoietic prostaglandin D synthase (H-PGDS; EC 5.3.99.2) produces prostaglandin D(2), an allergic and inflammatory mediator, in mast cells and Th2 cells. H-PGDS has been crystallized with novel inhibitors with half-maximal inhibitory concentrations (IC(50)) in the low nanomolar range by the counter-diffusion method onboard the Russian Service Module on the International Space Station. The X-ray diffraction of a microgravity-grown crystal of H-PGDS complexed with an inhibitor with an IC(50) value of 50 nM extended to 1.1 A resolution at 100 K using SPring-8 synchrotron radiation, which is one of the highest resolutions obtained to date for this protein.
Asunto(s)
Inhibidores Enzimáticos/química , Oxidorreductasas Intramoleculares/química , Lipocalinas/química , Cristalización , Cristalografía por Rayos X , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Lipocalinas/antagonistas & inhibidoresRESUMEN
A large high-quality crystal is required to specify the positions of H atoms in neutron structural analysis. Consequently, several methods have been proposed for obtaining such large crystals, and theoretical considerations for growing them have been presented. However, further investigation is required to obtain a numerical model that can provide quantitative experimental conditions for obtaining a single large crystal. In the case of protein crystallization experiments, the amount of sample is often limited. Therefore, it is more realistic to make a rough estimation from a small number of experiments. This paper proposes a method of estimating the optimum experimental conditions for the growth of large protein crystals by performing a small number of experiments using a micro-batch method and reporting a numerical model based on nucleation theory and a linear approximation of the crystal-growth rate. Specifically, micro-batch experiments are performed to provide the empirical parameters for the model and to help to estimate the conditions for the growth of a crystal of a predetermined size using a certain sample concentration and volume. This method is offered as a step on the path towards efficiently and rationally producing large crystals that can be subjected to neutron diffraction without depending on luck or on performing many experiments. It is expected to contribute to drug design and the elucidation of protein molecular functions and mechanisms by obtaining positional information on H atoms in the protein molecule, which is an advantage of neutron diffraction.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Proteínas/química , Cristalografía por Rayos X/métodosRESUMEN
We have developed a handling-free mounting method for X-ray crystallography of protein crystals at room temperatures-the glass capillary method. In this method, crystals were nucleated and grown on the capillary walls, and then, growth solutions were gently removed. The procedures for collecting data on the crystals were conducted by simply setting the capillary on the goniometer of a synchrotron beamline without touching the crystals. Crystal quality was characterized using mosaicity, resolution at I/σ(I) = 2, I/σ(I) at resolution = 2.0 Å, Rmerge, and completeness. Wilson plots were also used to characterize the quality of crystals. In particular, all samples showed very low mosaicity; the handling-free method successfully retained their low mosaicity and effectively maintained the crystal quality.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Cristalografía por Rayos X/instrumentación , Sincrotrones , TemperaturaRESUMEN
Antimicrobial resistance is a global public threat and raises the need for development of new antibiotics with a novel mode of action. The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to a new class of serine peptidases, family S46. Because S46 peptidases are not found in mammals, these enzymes are attractive targets for novel antibiotics. However, potent and selective inhibitors of these peptidases have not been developed to date. In this study, a high-resolution crystal structure analysis of PgDPP11 using a space-grown crystal enabled us to identify the binding of citrate ion, which could be regarded as a lead fragment mimicking the binding of a substrate peptide with acidic amino acids, in the S1 subsite. The citrate-based pharmacophore was utilized for in silico inhibitor screening. The screening resulted in an active compound SH-5, the first nonpeptidyl inhibitor of S46 peptidases. SH-5 and a lipophilic analog of SH-5 showed a dose-dependent inhibitory effect against the growth of P. gingivalis. The binding mode of SH-5 was confirmed by crystal structure analysis. Thus, these compounds could be lead structures for the development of selective inhibitors of PgDPP11.
Asunto(s)
Benzoatos/farmacología , Ácido Cítrico/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Porphyromonas gingivalis/enzimología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Benzoatos/química , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Cristalografía por Rayos X , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Fosfatos de Inositol , Modelos Moleculares , Conformación ProteicaRESUMEN
Old yellow enzyme (OYE) is an NADPH oxidoreductase that contains a flavin mononucleotide as a prosthetic group. The OYE from Trypanosoma cruzi, which produces prostaglandin F(2alpha), a potent mediator of various physiological and pathological processes, from prostaglandin H2. The protein was recombinantly expressed and purified from Escherichia coli and was crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 56.3, b = 78.8, c = 78.8 A, beta = 93.4 degrees and two molecules per asymmetric unit. The crystals were suitable for X-ray crystallographic studies and diffracted to 1.70 A resolution. A Patterson search method is in progress using the structure of OYE from Pseudomonas putida as a starting model.