RESUMO
Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.
Assuntos
Cistinil Aminopeptidase , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/metabolismo , Humanos , Cristalografia por Raios X , Especificidade por Substrato , Pirimidinas/química , Pirimidinas/farmacologia , Modelos Moleculares , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Conformação ProteicaRESUMO
Hypertrophic cardiomyopathy (HCM) is an inherited disease of the sarcomere resulting in excessive cardiac contractility. The first-in-class cardiac myosin inhibitor, mavacamten, improves symptoms in obstructive HCM. Here we present aficamten, a selective small-molecule inhibitor of cardiac myosin that diminishes ATPase activity by strongly slowing phosphate release, stabilizing a weak actin-binding state. Binding to an allosteric site on the myosin catalytic domain distinct from mavacamten, aficamten prevents the conformational changes necessary to enter the strongly actin-bound force-generating state. In doing so, aficamten reduces the number of functional myosin heads driving sarcomere shortening. The crystal structure of aficamten bound to cardiac myosin in the pre-powerstroke state provides a basis for understanding its selectivity over smooth and fast skeletal muscle. Furthermore, in cardiac myocytes and in mice bearing the hypertrophic R403Q cardiac myosin mutation, aficamten reduces cardiac contractility. Our findings suggest aficamten holds promise as a therapy for HCM.
Assuntos
Miosinas Cardíacas , Cardiomiopatia Hipertrófica , Contração Miocárdica , Animais , Cardiomiopatia Hipertrófica/tratamento farmacológico , Cardiomiopatia Hipertrófica/metabolismo , Humanos , Contração Miocárdica/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/genética , Modelos Animais de Doenças , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Camundongos , Cristalografia por Raios X , Mutação , Sarcômeros/metabolismo , Sarcômeros/efeitos dos fármacos , Actinas/metabolismo , Modelos Moleculares , Camundongos Transgênicos , Conformação ProteicaRESUMO
Identifying and characterizing metal-binding sites (MBS) within macromolecular structures is imperative for elucidating their biological functions. CheckMyMetal (CMM) is a web based tool that facilitates the interactive validation of MBS in structures determined through X-ray crystallography and cryo-electron microscopy (cryo-EM). Recent updates to CMM have significantly enhanced its capability to efficiently handle large datasets generated from cryo-EM structural analyses. In this study, we address various challenges inherent in validating MBS within both X-ray and cryo-EM structures. Specifically, we examine the difficulties associated with accurately identifying metals and modeling their coordination environments by considering the ongoing reproducibility challenges in structural biology and the critical importance of well annotated, high-quality experimental data. CMM employs a sophisticated framework of rules rooted in the valence bond theory for MBS validation. We explore how CMM validation parameters correlate with the resolution of experimentally derived structures of macromolecules and their complexes. Additionally, we showcase the practical utility of CMM by analyzing a representative cryo-EM structure. Through a comprehensive examination of experimental data, we demonstrate the capability of CMM to advance MBS characterization and identify potential instances of metal misassignment.
Assuntos
Microscopia Crioeletrônica , Metais , Microscopia Crioeletrônica/métodos , Sítios de Ligação , Cristalografia por Raios X/métodos , Metais/química , Metais/metabolismo , Reprodutibilidade dos Testes , Modelos Moleculares , Software , Substâncias Macromoleculares/químicaRESUMO
Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1ß. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.
Assuntos
Síndrome da Liberação de Citocina , Interleucina-6 , Humanos , Síndrome da Liberação de Citocina/tratamento farmacológico , Interleucina-6/metabolismo , Interleucina-6/antagonistas & inibidores , Cristalografia por Raios X , Receptores de Interleucina-6/antagonistas & inibidores , Receptores de Interleucina-6/metabolismo , Interleucina-1/metabolismo , Interleucina-1/antagonistas & inibidores , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/química , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Desenho de Fármacos , Receptor gp130 de Citocina/metabolismo , Receptor gp130 de Citocina/antagonistas & inibidores , Receptor gp130 de Citocina/química , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Receptores Tipo I de Interleucina-1/antagonistas & inibidores , Receptores Tipo I de Interleucina-1/metabolismoRESUMO
The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.
Assuntos
DNA , Quadruplex G , Insulina , Regiões Promotoras Genéticas , Insulina/química , Insulina/genética , DNA/química , DNA/genética , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Cristalografia por Raios X , Polimorfismo Genético , Sequências de Repetição em Tandem/genética , Sequência de Bases , Modelos Moleculares , Animais , Genes ReporterRESUMO
The otopetrin (OTOP) proteins were recently characterized as extracellular proton-activated proton channels. Several recent OTOP channel structures demonstrated that the channels form a dimer with each subunit adopting a double-barrel architecture. However, the structural mechanisms underlying some basic functional properties of the OTOP channels remain unresolved, including extracellular pH activation, proton conducting pathway, and rapid desensitization. In this study, we performed structural and functional characterization of the Caenorhabditis elegans OTOP8 (CeOTOP8) and mouse OTOP2 (mOTOP2) and illuminated a set of conformational changes related to the proton-conducting process in OTOP. The structures of CeOTOP8 reveal the conformational change at the N-terminal part of TM12 that renders the channel in a transiently proton-transferring state, elucidating an inter-barrel, Glu/His-bridged proton passage within each subunit. The structures of mOTOP2 reveal the conformational change at the N-terminal part of TM6 that exposes the central glutamate to the extracellular solution for protonation. In addition, the structural comparison between CeOTOP8 and mOTOP2, along with the structure-based mutagenesis, demonstrates that an inter-subunit movement at the OTOP channel dimer interface plays a central role in regulating channel activity. Combining the structural information from both channels, we propose a working model describing the multi-step conformational changes during the proton conducting process.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Canais Iônicos , Prótons , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Camundongos , Canais Iônicos/metabolismo , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Conformação Proteica , Concentração de Íons de Hidrogênio , Cristalografia por Raios X , Multimerização ProteicaRESUMO
To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.
Assuntos
Proteínas de Bactérias , Chlamydophila pneumoniae , Endocitose , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteína cdc42 de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Chlamydophila pneumoniae/metabolismo , Cristalografia por Raios X , Células HeLa , Interações Hospedeiro-Patógeno , Mimetismo Molecular , Ligação Proteica , Nexinas de Classificação/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , RatosRESUMO
Finding out about sample preparation and transportation of structural biology samples in Acta Crystallographica F, Structural Biology Communications.
Assuntos
Manejo de Espécimes , Cristalografia por Raios X , Manejo de Espécimes/métodos , HumanosRESUMO
12-oxophytodienoate reductase 3 (OPR3) is a key enzyme in the biosynthesis of jasmonoyl-L-isoleucine, the receptor-active form of jasmonic acid and crucial signaling molecule in plant defense. OPR3 was initially crystallized as a self-inhibitory dimer, implying that homodimerization regulates enzymatic activity in response to biotic and abiotic stresses. Since a sulfate ion is bound to Y364, mimicking a phosphorylated tyrosine, it was suggested that dimer formation might be controlled by reversible phosphorylation of Y364 in vivo. To investigate OPR3 homodimerization and its potential physiological role in more detail, we performed analytical gel filtration and dynamic light scattering on wild-type OPR3 and three variants (R283D, R283E, and Y364P). The experiments revealed a rapid and highly sensitive monomer-dimer equilibrium for all OPR3 constructs. We crystallized all constructs with and without sulfate to examine its effect on the dimerization process and whether reversible phosphorylation of Y364 triggers homodimerization in vivo. All OPR3 constructs crystallized in their monomeric and dimeric forms independent of the presence of sulfate. Even variant Y364P, lacking the putative phosphorylation site, was crystallized as a self-inhibitory homodimer, indicating that Y364 is not required for dimerization. Generally, the homodimer is relatively weak, and our results raise doubts about its physiological role in regulating jasmonate biosynthesis.
Assuntos
Multimerização Proteica , Fosforilação , Oxilipinas/metabolismo , Ciclopentanos/metabolismo , Oxirredutases/metabolismo , Oxirredutases/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/química , Cristalografia por Raios X , Solanum lycopersicum/metabolismo , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Sulfatos/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CHRESUMO
Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism.
Assuntos
Acinetobacter baumannii , Proteínas de Bactérias , Grupo dos Citocromos b , Ferritinas , Ferritinas/química , Ferritinas/metabolismo , Acinetobacter baumannii/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Multimerização Proteica , Ferro/metabolismo , Ferro/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Oxirredução , Conformação ProteicaRESUMO
A zinc metallopeptidase neurolysin (Nln) processes diverse bioactive peptides to regulate signaling in the mammalian nervous system. To understand how Nln interacts with various peptides with dissimilar sequences, we determined crystal structures of Nln in complex with diverse peptides including dynorphins, angiotensin, neurotensin, and bradykinin. The structures show that Nln binds these peptides in a large dumbbell-shaped interior cavity constricted at the active site, making minimal structural changes to accommodate different peptide sequences. The structures also show that Nln readily binds similar peptides with distinct registers, which can determine whether the peptide serves as a substrate or a competitive inhibitor. We analyzed the activities and binding of Nln toward various forms of dynorphin A peptides, which highlights the promiscuous nature of peptide binding and shows how dynorphin A (1-13) potently inhibits the Nln activity while dynorphin A (1-8) is efficiently cleaved. Our work provides insights into the broad substrate specificity of Nln and may aid in the future design of small molecule modulators for Nln.
Assuntos
Dinorfinas , Neurotensina , Humanos , Especificidade por Substrato , Dinorfinas/química , Dinorfinas/metabolismo , Neurotensina/química , Neurotensina/metabolismo , Metaloendopeptidases/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/antagonistas & inibidores , Ligação Proteica , Cristalografia por Raios X , Modelos Moleculares , Domínio Catalítico , Bradicinina/química , Bradicinina/metabolismo , Angiotensinas/metabolismo , Angiotensinas/química , Sequência de AminoácidosRESUMO
We have determined the crystal structure of a pseudoknot (PK)-containing hammerhead ribozyme that closely resembles the pistol ribozyme, with essentially identical secondary structure and connectivity. The activity is more sensitive to deletion of the G8 2'OH than to the absence of magnesium ions, indicating that the catalytic mechanism is the same as the extended hammerhead, and distinct from the pistol ribozyme. Here we show that nucleophilic attack is almost perfectly in-line, and the G8 2'OH is well positioned to act as general acid, being directed towards the O5' leaving group, and 2.9 Å away from it. Despite the similarity in overall structure to the pistol ribozyme, the local structure close to the cleavage site differs, and the PK hammerhead retains its unique mechanistic identity and demonstrates enhanced activity over other hammerhead ribozymes under standard conditions.
Assuntos
Conformação de Ácido Nucleico , RNA Catalítico , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA Catalítico/genética , Catálise , Cristalografia por Raios X , Modelos Moleculares , Magnésio/metabolismo , Magnésio/químicaRESUMO
Lappaconitine, a diterpene alkaloid isolated from Aconitum sinomontanum Nakai, exhibits a wide range of biological activities, making it a promising candidate for the development of novel derivatives with therapeutic potential. In our research, we executed a two-step transformation via oxidative cleavage of lappaconitine's vicinal diol using the hypervalent iodine reagent PhI(OAc)2, followed by strong alkaline hydrolysis. This approach yielded four new unanticipated compounds, whose structures were identified by spectroscopic methods and/or X-ray crystallography. Thus, we proposed plausible reaction mechanisms for their formations and particularly investigated the remarkable diastereoselectivity for the formation of single stereoisomer 8 observed during the alkaline hydrolysis step. Among them, compound 8 (code name: QG3030) demonstrated both enhanced osteogenic differentiation of human mesenchymal stem cells and significant osteogenic effect in an ovariectomized rat model with no acute oral toxicity.
Assuntos
Aconitina , Iodo , Aconitina/análogos & derivados , Aconitina/química , Aconitina/farmacologia , Humanos , Animais , Estrutura Molecular , Ratos , Iodo/química , Alcaloides/química , Alcaloides/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Aconitum/química , Cristalografia por Raios X , Osteogênese/efeitos dos fármacos , Estereoisomerismo , Diferenciação Celular/efeitos dos fármacosRESUMO
African swine fever virus (ASFV) is the causal agent of African swine fever (ASF), which is contagious and highly lethal to domestic pigs and wild boars. The genome of ASFV encodes many proteins important for ASFV life cycle. The functional importance of topoisomerase AsfvTopII has been confirmed by in vivo and in vitro assays, but the structure of AsfvTopII is poorly studied. Here, we report four AsfvTopII complex structures. The ATPase domain structures reveal the detailed basis for ATP binding and hydrolysis, which is shared by AsfvTopII and eukaryotic TopIIs. The DNA-bound structures show that AsfvTopII follows conserved mechanism in G-DNA binding and cleavage. Besides G-DNA, a T-DNA fragment is also captured in one AsfvTopII structure. Mutagenesis and in vitro assays confirm that Pro852 and the T-DNA-binding residue Tyr744 are important for the function of AsfvTopII. Our study not only advances the understanding on the biological function of AsfvTopII, but also provides a solid basis for the development of AsfvTopII-specific inhibitors.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Proteínas Virais , Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/enzimologia , Animais , Suínos , Febre Suína Africana/virologia , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/química , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Ligação Proteica , DNA Viral/genética , DNA Viral/metabolismo , Cristalografia por Raios XRESUMO
Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Hidrolases de Éster Carboxílico , Lactonas , Arabidopsis/metabolismo , Arabidopsis/enzimologia , Lactonas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/genética , Cristalografia por Raios X , Reguladores de Crescimento de Plantas/metabolismo , Modelos Moleculares , Hidrólise , Conformação ProteicaRESUMO
The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and ß-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded ß-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the ß-flap inducing the active conformation of catalytic residues. The ß-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δß-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the ß-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the ß-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.
Assuntos
Ácido Fítico , Vibrio vulnificus , Vibrio vulnificus/enzimologia , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Ácido Fítico/metabolismo , Domínio Catalítico , Cisteína Proteases/metabolismo , Cisteína Proteases/química , Cisteína Proteases/genética , Cristalografia por Raios X , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Domínios Proteicos , Modelos Moleculares , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sequência de AminoácidosRESUMO
Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.
Assuntos
Hexaclorocicloexano , Hidrolases , Hidrolases/metabolismo , Hidrolases/genética , Hidrolases/química , Hexaclorocicloexano/metabolismo , Especificidade por Substrato , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Cinética , Biodegradação Ambiental , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Sphingomonas/enzimologia , Sphingomonas/genética , Sphingomonas/metabolismoRESUMO
Twelve spiro thiazolidinone compounds (A-L) were synthesized via either conventional thermal or ultrasonication techniques using Fe2O3 nanoparticles. The modification of the traditional procedure by using Fe2O3 nanoparticles led to enhancement of the yield of the desired candidates to 78-93% in approximately half reaction time compared with 58-79% without catalyst. The products were fully characterized using different analytical and spectroscopic techniques. The structure of the two derivatives 4-phenyl-1-thia-4-azaspirodecan-3-one (A) and 4-(p-tolyl)-1-thia-4-azaspirodecan-3-one (B) were also determined using single crystal X-ray diffraction and Hirshfeld surface analysis. The two compounds (A and B) were crystallized in the orthorhombic system with Pbca and P212121 space groups, respectively. In addition, the crystal packing of compounds revealed the formation of supramolecular array with a net of intermolecular hydrogen bonding interactions. The energy optimized geometries of some selected derivatives were performed by density functional theory (DFT/B3LYP). The reactivity descriptors were also calculated and correlated with their biological properties. All the reported compounds were screened for antimicrobial inhibitions. The two derivatives, F and J, exhibited the highest levels of bacterial inhibition with an inhibition zone of 10-17 mm. Also, the two derivatives, F and J, displayed the most potent fungal inhibition with an inhibition zone of 15-23 mm. Molecular docking investigations of some selected derivatives were performed using a B-DNA (PDB: 1BNA) as a macromolecular target. Structure and activity relationship of the reported compounds were correlated with the data of antimicrobial activities and the computed reactivity parameters.
Assuntos
Simulação de Acoplamento Molecular , Tiazolidinas , Catálise , Tiazolidinas/química , Tiazolidinas/síntese química , Tiazolidinas/farmacologia , Compostos de Espiro/química , Compostos de Espiro/síntese química , Compostos de Espiro/farmacologia , Cristalografia por Raios X/métodos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Teoria da Densidade Funcional , Testes de Sensibilidade Microbiana , Compostos Férricos/química , Ligação de HidrogênioRESUMO
Building blocks have been identified that can be functionalised by sequential nucleophilic aromatic substitution. Some examples are reported that involve the formation of cyclic benzodioxin and phenoxathiine derivatives from 4,5-difluoro-1,2-dinitrobenzene, racemic quinoxaline thioethers, and sulfones from 2,3-dichloroquinoxaline and (2-aminophenylethane)-2,5-dithiophenyl-4-nitrobenzene from 1-(2-aminophenylethane)-2-fluoro-4,5-dinitrobenzene. Four X-ray single-crystal structure determinations are reported, two of which show short intermolecular N-O N "π hole" contacts.
Assuntos
Halogênios , Cristalografia por Raios X , Halogênios/química , Estrutura Molecular , Modelos Moleculares , Quinoxalinas/químicaRESUMO
Glucagon receptor (GCGR) is a class B1 G-protein-coupled receptor that plays a crucial role in maintaining human blood glucose homeostasis and is a significant target for the treatment of type 2 diabetes mellitus (T2DM). Currently, six small molecules (Bay 27-9955, MK-0893, MK-3577, LY2409021, PF-06291874, and LGD-6972) have been tested or are undergoing clinical trials, but only the binding site of MK-0893 has been resolved. To predict binding sites for other small molecules, we utilized both the crystal structure of the GCGR and MK-0893 complex and dynamic conformations. We docked five small molecules and selected the best conformation based on binding mode, docking score, and binding free energy. We performed MD simulations to verify the binding mode of the selected small molecules. Moreover, when selecting conformations, results of competitive binding were referred to. MD simulation indicated that Bay 27-9955 exhibits moderate binding stability in Pocket 3. MK-3577, LY2409021, and PF-06291874 exhibited highly stable binding to Pocket 2, consistent with experimental results. However, LY2409021 may also bind to Pocket 5. Additionally, LGD-6972 exhibited relatively stable binding in Pocket 5. We also conducted structural modifications of LGD-6972 based on the results of MD simulations and predicted its analogues' bioavailability, providing a reference for the study of GCGR small molecules.