RESUMO
Nicotinamide-adenine dinucleotide (NAD) is centrally important to metabolic reactions that involve redox chemistry. In bacteria, NAD biosynthesis is controlled by different transcription factors, depending on the species. Among the four regulators identified so far, the protein NadQ is reported to act as a repressor of the de novo NAD biosynthetic pathway in proteobacteria. Using comparative genomics, a systematic reconstruction of NadQ regulons in thousands of fully sequenced bacterial genomes has been performed, confirming that NadQ is present in α-proteobacteria and some ß- and γ-proteobacteria, including pathogens like Bordetella pertussis and Neisseria meningitidis, where it likely controls de novo NAD biosynthesis. Through mobility shift assay and mutagenesis, the DNA binding activity of NadQ from Agrobacterium tumefaciens was experimentally validated and determined to be suppressed by ATP. The crystal structures of NadQ in native form and in complex with ATP were determined, indicating that NadQ is a dimer, with each monomer composed of an N-terminal Nudix domain hosting the effector binding site and a C-terminal winged helix-turn-helix domain that binds DNA. Within the dimer, we found one ATP molecule bound, at saturating concentration of the ligand, in keeping with an intrinsic asymmetry of the quaternary structure. Overall, this study provided the basis for depicting a working model of NadQ regulation mechanism.
Assuntos
Bactérias , NAD , Trifosfato de AdenosinaRESUMO
Invited for the cover of this issue is the collaborative research team coordinated by Arie vanâ derâ Lee at the University of Montpellier. The image depicts chiral channels with highly mobile water molecules resulting from the robust self-organization of a simple achiral acetamide. Fully reversible release and re-uptake of water molecules takes place near ambient conditions, with efficient water transport and a good selectivity against NaCl suggesting it to be an efficient candidate for desalination processes. Read the full text of the article at 10.1002/chem.20200383.
Assuntos
Aquaporinas , Água , AcetamidasRESUMO
Achiral 2-hydroxy-N-(diphenylmethyl)acetamide (HNDPA) crystallizes in the P61 chiral space group as a hydrate, building up permeable chiral crystalline helical water channels. The crystallization-driven chiral self-resolution process is highly robust, with the same air-stable crystalline form readily obtained under a variety of conditions. Interestingly, the HNDPA supramolecular helix inner pore is filled by a helical water wire. The whole edifice is mainly stabilized by robust hydrogen bonds involving the HNDPA amide bonds and CH π interactions between the HNDPA phenyl groups. The crystalline structure shows breathing behavior, with completely reversible release and re-uptake of water inside the chiral channel under ambient conditions. Importantly, the HNDPA channel is able to transport water very efficiently and selectively under biomimetic conditions. With a permeability per channel of 3.3 million water molecules per second in large unilamellar vesicles (LUV) and total selectivity against NaCl, the HNDPA channel is a very promising functional nanomaterial for future applications.
Assuntos
Aquaporinas , Água , Acetamidas , Cristalização , Ligação de Hidrogênio , Água/químicaRESUMO
Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.
Assuntos
Dipeptídeos/metabolismo , Inibidores Enzimáticos/metabolismo , gama-Glutamiltransferase/antagonistas & inibidores , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dipeptídeos/química , Humanos , Modelos Moleculares , Conformação Proteica , gama-Glutamiltransferase/química , gama-Glutamiltransferase/metabolismoRESUMO
The RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni2+ , Mn2+ , Mg2+ , Fe2+ , and Zn2+ . While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn2+ ion), recent structures determined a Zn2+ /Fe2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn2+ /Mn2+ occupancy. ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.
Assuntos
Biocatálise , Entamoeba histolytica/enzimologia , Manganês/metabolismo , RNA Nucleotidiltransferases/química , RNA Nucleotidiltransferases/metabolismo , Zinco/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos MolecularesRESUMO
Intense efforts are underway to identify inhibitors of the enzyme gamma-glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma-glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6-diazo-5-oxo-norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM-1 min-1 and the Ki was 2.7 ± 0.7 mM. The crystal structure of DON-inactivated hGGT1 contained a molecule of DON without the diazo-nitrogen atoms in the active site. The overall structure of the hGGT1-DON complex resembled the structure of the apo-enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1-DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α-amine of Thr381. The structure of DON-bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.
Assuntos
Diazo-Oxo-Norleucina/química , Inibidores Enzimáticos/química , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/química , Domínio Catalítico , Cristalografia por Raios X , Humanos , Estrutura Secundária de ProteínaRESUMO
The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.
Assuntos
Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , Formaldeído/química , Histonas/química , Histonas/genética , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , UbiquitinaçãoRESUMO
Shroom-mediated remodeling of the actomyosin cytoskeleton is a critical driver of cellular shape and tissue morphology that underlies the development of many tissues including the neural tube, eye, intestines, and vasculature. Shroom uses a conserved SD2 domain to direct the subcellular localization of Rho-associated kinase (Rock), which in turn drives changes in the cytoskeleton and cellular morphology through its ability to phosphorylate and activate non-muscle myosin II. Here, we present the structure of the human Shroom-Rock binding module, revealing an unexpected stoichiometry for Shroom in which two Shroom SD2 domains bind independent surfaces on Rock. Mutation of interfacial residues impaired Shroom-Rock binding in vitro and resulted in altered remodeling of the cytoskeleton and loss of Shroom-mediated changes in cellular morphology. Additionally, we provide the first direct evidence that Shroom can function as a Rock activator. These data provide molecular insight into the Shroom-Rock interface and demonstrate that Shroom directly participates in regulating cytoskeletal dynamics, adding to its known role in Rock localization.
Assuntos
Receptor Quinase 1 Acoplada a Proteína G/química , Proteínas de Membrana/química , Proteínas dos Microfilamentos/química , Complexos Multiproteicos/química , Receptor Quinase 1 Acoplada a Proteína G/genética , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Miosina Tipo II/química , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Triosephosphate isomerase (TPI) deficiency is a poorly understood disease characterized by hemolytic anemia, cardiomyopathy, neurologic dysfunction, and early death. TPI deficiency is one of a group of diseases known as glycolytic enzymopathies, but is unique for its severe patient neuropathology and early mortality. The disease is caused by missense mutations and dysfunction in the glycolytic enzyme, TPI. Previous studies have detailed structural and catalytic changes elicited by disease-associated TPI substitutions, and samples of patient erythrocytes have yielded insight into patient hemolytic anemia; however, the neuropathophysiology of this disease remains a mystery. This study combines structural, biochemical, and genetic approaches to demonstrate that perturbations of the TPI dimer interface are sufficient to elicit TPI deficiency neuropathogenesis. The present study demonstrates that neurologic dysfunction resulting from TPI deficiency is characterized by synaptic vesicle dysfunction, and can be attenuated with catalytically inactive TPI. Collectively, our findings are the first to identify, to our knowledge, a functional synaptic defect in TPI deficiency derived from molecular changes in the TPI dimer interface.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/genética , Erros Inatos do Metabolismo dos Carboidratos/genética , Drosophila melanogaster/genética , Doenças do Sistema Nervoso/genética , Vesículas Sinápticas/genética , Triose-Fosfato Isomerase/deficiência , Triose-Fosfato Isomerase/genética , Anemia Hemolítica Congênita não Esferocítica/patologia , Animais , Comportamento Animal , Erros Inatos do Metabolismo dos Carboidratos/patologia , Cristalografia por Raios X , Dimerização , Humanos , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/patologia , Conformação Proteica , Vesículas Sinápticas/patologia , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/metabolismoRESUMO
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
Assuntos
Cristalografia por Raios X/instrumentação , Enzimas/química , Acústica , Cristalografia por Raios X/métodos , Modelos Moleculares , Muramidase/química , Conformação Proteica , Termolisina/químicaRESUMO
Improved understanding of the relationship among structure, dynamics, and function for the enzyme phenylalanine hydroxylase (PAH) can lead to needed new therapies for phenylketonuria, the most common inborn error of amino acid metabolism. PAH is a multidomain homo-multimeric protein whose conformation and multimerization properties respond to allosteric activation by the substrate phenylalanine (Phe); the allosteric regulation is necessary to maintain Phe below neurotoxic levels. A recently introduced model for allosteric regulation of PAH involves major domain motions and architecturally distinct PAH tetramers [Jaffe EK, Stith L, Lawrence SH, Andrake M, Dunbrack RL, Jr (2013) Arch Biochem Biophys 530(2):73-82]. Herein, we present, to our knowledge, the first X-ray crystal structure for a full-length mammalian (rat) PAH in an autoinhibited conformation. Chromatographic isolation of a monodisperse tetrameric PAH, in the absence of Phe, facilitated determination of the 2.9 Å crystal structure. The structure of full-length PAH supersedes a composite homology model that had been used extensively to rationalize phenylketonuria genotype-phenotype relationships. Small-angle X-ray scattering (SAXS) confirms that this tetramer, which dominates in the absence of Phe, is different from a Phe-stabilized allosterically activated PAH tetramer. The lack of structural detail for activated PAH remains a barrier to complete understanding of phenylketonuria genotype-phenotype relationships. Nevertheless, the use of SAXS and X-ray crystallography together to inspect PAH structure provides, to our knowledge, the first complete view of the enzyme in a tetrameric form that was not possible with prior partial crystal structures, and facilitates interpretation of a wealth of biochemical and structural data that was hitherto impossible to evaluate.
Assuntos
Biopolímeros/química , Fenilalanina Hidroxilase/química , Animais , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , RatosRESUMO
La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.
Assuntos
Regiões 5' não Traduzidas , Autoantígenos/química , Ribonucleoproteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Autoantígenos/metabolismo , Sequência Conservada , Sequências Hélice-Volta-Hélice , Humanos , Modelos Moleculares , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos , Ribonucleoproteínas/metabolismo , Eletricidade Estática , Antígeno SS-BRESUMO
Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5'-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM-small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.
Assuntos
Antibacterianos/química , Transferases Intramoleculares/química , Simulação de Acoplamento Molecular , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Transferases Intramoleculares/genética , Ligantes , Modelos Biológicos , Estrutura MolecularRESUMO
Interchanging Leu-119 for Pro-119 at the tip of the ß4-ß5 loop in the first FK506 binding domain (FK1) of the FKBP51 and FKBP52 proteins, respectively, has been reported to largely reverse the inhibitory (FKBP51) or stimulatory (FKBP52) effects of these co-chaperones on the transcriptional activity of glucocorticoid and androgen receptor-protein complexes. Previous NMR relaxation studies have identified exchange line broadening, indicative of submillisecond conformational motion, throughout the ß4-ß5 loop in the FK1 domain of FKBP51, which are suppressed by the FKBP52-like L119P substitution. This substitution also attenuates exchange line broadening in the underlying ß2 and ß3a strands that is centered near a bifurcated main chain hydrogen bond interaction between these two strands. The present study demonstrates that these exchange line broadening effects arise from two distinct coupled conformational transitions, and the transition within the ß2 and ß3a strands samples a transient conformation that resembles the crystal structures of the selectively inhibited FK1 domain of FKBP51 recently reported. Although the crystal structures for their series of inhibitors were interpreted as evidence for an induced fit mechanism of association, the presence of a similar conformation being significantly populated in the unliganded FKBP51 domain is more consistent with a conformational selection binding process. The contrastingly reduced conformational plasticity of the corresponding FK1 domain of FKBP52 is consistent with the current model in which FKBP51 binds to both the apo- and hormone-bound forms of the steroid receptor to modulate its affinity for ligand, whereas FKBP52 binds selectively to the latter state.
Assuntos
Modelos Moleculares , Receptores Androgênicos/química , Receptores de Glucocorticoides/química , Proteínas de Ligação a Tacrolimo/química , Substituição de Aminoácidos , Humanos , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.
Assuntos
gama-Glutamiltransferase/química , Aminobutiratos/química , Aminobutiratos/farmacologia , Apoenzimas/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/metabolismo , Humanos , Modelos Moleculares , Organofosfonatos/química , Organofosfonatos/farmacologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , gama-Glutamiltransferase/antagonistas & inibidores , gama-Glutamiltransferase/genéticaRESUMO
This paper uses the binding pocket of human carbonic anhydrase II (HCAII, EC 4.2.1.1) as a tool to examine the properties of Hofmeister anions that determine (i) where, and how strongly, they associate with concavities on the surfaces of proteins and (ii) how, upon binding, they alter the structure of water within those concavities. Results from X-ray crystallography and isothermal titration calorimetry show that most anions associate with the binding pocket of HCAII by forming inner-sphere ion pairs with the Zn(2+) cofactor. In these ion pairs, the free energy of anion-Zn(2+) association is inversely proportional to the free energetic cost of anion dehydration; this relationship is consistent with the mechanism of ion pair formation suggested by the "law of matching water affinities". Iodide and bromide anions also associate with a hydrophobic declivity in the wall of the binding pocket. Molecular dynamics simulations suggest that anions, upon associating with Zn(2+), trigger rearrangements of water that extend up to 8 Å away from their surfaces. These findings expand the range of interactions previously thought to occur between ions and proteins by suggesting that (i) weakly hydrated anions can bind complementarily shaped hydrophobic declivities, and that (ii) ion-induced rearrangements of water within protein concavities can (in contrast with similar rearrangements in bulk water) extend well beyond the first hydration shells of the ions that trigger them. This study paints a picture of Hofmeister anions as a set of structurally varied ligands that differ in size, shape, and affinity for water and, thus, in their ability to bind toand to alter the charge and hydration structure ofpolar, nonpolar, and topographically complex concavities on the surfaces of proteins.
Assuntos
Anidrase Carbônica II/metabolismo , Ânions , Sítios de Ligação , Anidrase Carbônica II/química , Coenzimas , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Termodinâmica , ZincoRESUMO
We report that in the red light-absorbing (Pr) state, the bilin chromophore of the Deinococcus radiodurans proteobacterial phytochrome (DrBphP) is hypersensitive to X-ray photons used in typical synchrotron X-ray protein crystallography experiments. This causes the otherwise fully protonated chromophore to deprotonate without additional major structural changes. These results have major implications for our understanding of the structural and chemical characteristics of the resting and intermediate states of phytochromes and other photoreceptor proteins.
Assuntos
Proteínas de Bactérias/química , Pigmentos Biliares/química , Deinococcus , Fitocromo/química , Prótons , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Conformação Proteica , Raios X/efeitos adversosRESUMO
Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI(I170V) elicits behavioral abnormalities in Drosophila. An examination of hTPI(I170V) enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis.
Assuntos
Anemia Hemolítica Congênita não Esferocítica/patologia , Erros Inatos do Metabolismo dos Carboidratos/patologia , Domínio Catalítico , Triose-Fosfato Isomerase/deficiência , Triose-Fosfato Isomerase/metabolismo , Anemia Hemolítica Congênita não Esferocítica/enzimologia , Animais , Comportamento Animal , Erros Inatos do Metabolismo dos Carboidratos/enzimologia , Modelos Animais de Doenças , Drosophila , Estabilidade Enzimática , Humanos , Mutação , Triose-Fosfato Isomerase/química , Triose-Fosfato Isomerase/genéticaRESUMO
In prokaryotes, RNA derived from type I and type III CRISPR loci direct large ribonucleoprotein complexes to destroy invading bacteriophage and plasmids. In Escherichia coli, this 405-kilodalton complex is called Cascade. We report the crystal structure of Cascade bound to a single-stranded DNA (ssDNA) target at a resolution of 3.03 angstroms. The structure reveals that the CRISPR RNA and target strands do not form a double helix but instead adopt an underwound ribbon-like structure. This noncanonical structure is facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid and is stabilized by the highly interlocked organization of protein subunits. These studies provide insight into both the assembly and the activity of this complex and suggest a mechanism to enforce fidelity of target binding.
Assuntos
Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , RNA Bacteriano/química , Cristalografia por Raios X , DNA Helicases/química , Modelos MolecularesRESUMO
Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource group. This mini-gap insertion-device beamline has seen constant upgrades for the last seven years in order to achieve mini-beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state-of-the-art facility for the scientific community.