RESUMO
The role of Alpha folate receptors (FRα) in folate metabolism and cancer development has been extensively studied. The reason for this is not only associated to its direct relation to disease development but also to its potential use as a highly sensitive and specific biomarker for cancers therapies. Over the recent years, the crystal structures of human FRα complexed with different ligands were described relying on an expensive and time-consuming production process. Here, we constructed an efficient system for the expression and purification of a human FRα in E. coli. Unlike a conventional expression method we used a specific protein fusion expressing the target protein together with a trigger factor (TF). This factor is a chaperone from E. coli that assists the correct folding of newly synthesized polypeptide chains. The activity of rTFFRα was comparable to glycosylphosphatidylinositol (GPI) anchored proteins extracted from HeLa tumor cells. Our work demonstrates a straightforward and versatile approach for the production of active human FRα by heterologous expression; this approach further enhances the development of inhibition studies and biotechnological applications. The purified product was then conjugated to liposomes, obtaining a 35% higher signal from densitometry measurement on the immunoblotting assay in the contruct containing the Ni-NTA tag, as a mimesis of an exosome, which is of vital importance to nanotherapeutic techniques associated to treatment and diagnosis of tumors.
Assuntos
Proteínas de Escherichia coli/genética , Receptor 1 de Folato/genética , Peptidilprolil Isomerase/genética , Plasmídeos/química , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Receptor 1 de Folato/metabolismo , Expressão Gênica , Células HeLa , Histidina/genética , Histidina/metabolismo , Humanos , Cinética , Lipossomos/química , Lipossomos/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptidilprolil Isomerase/metabolismo , Plasmídeos/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Tetra-Hidrofolatos/metabolismoRESUMO
BACKGROUND: The ZNF706 gene encodes a protein that belongs to the zinc finger family of proteins and was found to be highly expressed in laryngeal cancer, making the structure and function of ZNF706 worthy of investigation. In this study, we expressed and purified recombinant human ZNF706 that was suitable for structural analysis in Escherichia coli BL21(DH3). FINDINGS: ZNF706 mRNA was extracted from a larynx tissue sample, and cDNA was ligated into a cloning vector using the TOPO method. ZNF706 protein was expressed according to the E. coli expression system procedures and was purified using a nickel-affinity column. The structural qualities of recombinant ZNF706 and quantification alpha, beta sheet, and other structures were obtained by spectroscopy of circular dichroism. ZNF706's structural modeling showed that it is composed of α-helices (28.3%), ß-strands (19.4%), and turns (20.9%), in agreement with the spectral data from the dichroism analysis. CONCLUSIONS: We used circular dichroism and molecular modeling to examine the structure of ZNF706. The results suggest that human recombinant ZNF706 keeps its secondary structures and is appropriate for functional and structural studies. The method of expressing ZNF706 protein used in this study can be used to direct various functional and structural studies that will contribute to the understanding of its function as well as its relationship with other biological molecules and its putative role in carcinogenesis.
RESUMO
The enzyme Homoserine dehydrogenase from Paracoccidioides brasiliensis (PbHSD), an interesting enzyme in the search for new antifungal drugs against paracoccidioidomycosis, was expressed by E. coli. Thirty milligrams of PbHSD with 94% of purity were obtained per liter of culture medium. The analysis by CD spectroscopy indicates a composition of 45.5 ± 7.3% of α-helices and 10.5 ± 7.0% ß-strands. Gel filtration chromatography indicates a homodimer as biological unity. Fluorescence emission spectroscopy has shown stability of PbHSD in the presence of urea until Cm of 4.13 ± 0.21 M, and a broad pH range in which there is no conformational change. The protein analysis by differential scanning calorimetry indicates high stability at room temperature, but low stability at high temperatures, suffering irreversible denaturation, with Tm = 58.65 ± 0.87 °C. Kinetic studies of PbHSD by molecular absorption spectroscopy in UV/Vis have shown an optimum pH between 9.35 and 9.50, with Michaelian behavior, presenting KM of 224 ± 15 µM and specific activity at optimum pH of 2.10 ± 0.07 µmol/min/mg for homoserine. Therefore, protein expression and purification were efficient, and the structural characterization has shown that PbHSD presents native conformation with enzymatic activity in kinetic assays.
Assuntos
Paracoccidioides , Paracoccidioides/genética , Paracoccidioides/metabolismo , Homosserina Desidrogenase/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Espectrometria de FluorescênciaRESUMO
Cyclin-dependent kinases (CDKs) comprise a family of about 20 serine/threonine kinases whose catalytic activity requires a regulatory subunit known as cyclin; these enzymes play several roles in the cell cycle and transcription. PCTAIRE kinases (PCTKs) are a CDK subfamily, characterized by serine to cysteine mutation in the consensus PSTAIRE motif, involved in binding to the cyclin. One member of this class is PCTK3, which has two isoforms (a and b) and is also known as CDK18. After being activated by cyclin A2 or phosphorylation at Ser12 by PKA, PCTK3 can perform several functions. Among these functions, we may highlight the following: modulation of cargo transport in membrane traffic, p53-responsive gene, regulation of genome integrity. According to different studies, PCTK3 dysfunction is related to a wide range of diseases, such as metabolic diseases, cerebral ischemia, depression, cancer, neurological disorders, and Alzheimer's disease. Although this protein participates in different biological events, we may say that PCTK3 has received far less attention than other CDKs. There are thousands of published articles about other CDKs and less than two hundred articles related to PCTK3. The main objective of this review is to present the selected published studies about this protein. Our focus is on PCTK3 particularities compared to other CDKs. Here we give an overview of the biological functions of PCTK3 and explore its potential as a target for drug design.
Assuntos
Quinases Ciclina-Dependentes , Proteínas Serina-Treonina Quinases , Ciclo Celular , Pontos de Checagem do Ciclo Celular , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , FosforilaçãoRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by fungi of the genus Paracoccidioides spp., which mainly affects workers in rural regions of Latin America. Although the antifungal agents currently available for the treatment of PCM are effective in controlling the disease, many months are needed for healing, making the side effects and drug interactions relevant. In addition, conventional treatments are not able to control the sequelae left by PCM, even after the cure, justifying the search for new therapeutic options against PCM. In this context, the enzyme homoserine dehydrogenase of P. brasiliensis (PbHSD) was used to screen a library of natural products from the Zinc database using three different docking programs, i.e. Autodock, Molegro, and CLC Drugdiscovery Workbench. Three molecules (Zinc codes 2123137, 15967722, and 20611644) were better ranked than the homoserine substrate (HSE) and were used for in vitro trials of the minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MCF). All three molecules presented a fungicidal profile with MICs/MCFs of 8, 32, and 128 µg mL-1, respectively. The two most promising molecules presented satisfactory results with wide therapeutic ranges in the cytotoxicity assays. Molecular dynamics simulations of PbHSD indicated that the ligands remained bound to the protein by a common mechanism throughout the simulation. The molecule with the lowest MIC value presented the highest number of contacts with the protein. The results presented in this work suggest that the molecule Zinc2123137 may be considered as a hit in the development of new therapeutic options for PCM.
Assuntos
Antifúngicos/farmacologia , Homosserina Desidrogenase/antagonistas & inibidores , Paracoccidioides/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Células HeLa , Humanos , Ligantes , Testes de Sensibilidade Microbiana/métodos , Simulação de Dinâmica Molecular , Células VeroRESUMO
AIM: Novel 4-methoxy-naphthalene derivatives were synthesized based on hits structures in order to evaluate the antifungal activity against Paracoccidioides spp. METHODS: Antifungal activity of compounds was evaluated against P. brasiliensis and most promising compounds 2 and 3 were tested against eight clinically important fungal species. RESULTS: Compound 3 was the more active compound with MIC 8 to 32 µg.ml-1 for Paracoccidioides spp without toxicity monkey kidney and murine macrophagecells. Carbohydrazide 3 showed good synergistic antifungal activity with amphotericin B against P. brasiliensis specie. Titration assay of carbohydrazide 3 with PbHSD enzyme demonstrates the binding ligand-protein. Molecular dynamics simulations show that ligand 3 let the PbHSD protein more stable. CONCLUSION: New carbohydrazide 3 is an attractive lead for drug development to treat paracoccidioidomycoses.
Assuntos
Antifúngicos/farmacologia , Naftalenos/farmacologia , Paracoccidioides/efeitos dos fármacos , Paracoccidioidomicose/tratamento farmacológico , Anfotericina B/farmacologia , Animais , Antifúngicos/uso terapêutico , Chlorocebus aethiops , Combinação de Medicamentos , Sinergismo Farmacológico , Homosserina Desidrogenase/metabolismo , Hidrazinas/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Naftalenos/síntese química , Naftalenos/uso terapêutico , Paracoccidioides/patogenicidade , Estabilidade Proteica , Células Vero/efeitos dos fármacosRESUMO
The family of Cyclin-Dependent Kinases (CDKs) can be subdivided into two major functional groups based on their roles in cell cycle and/or transcriptional control. CDK9 is the catalytic subunit of positive transcription elongation factor b (P-TEFb). CDK9 is the kinase of the TAK complex (Tat-associated kinase complex), and binds to Tat protein of HIV, suggesting a possible role for CDK9 in AIDS progression. CDK9 complexed with its regulatory partner cyclin T1, serves as a cellular mediator of the transactivation function of the HIV Tat protein. P-TEFb is responsible for the phosphorylation of the carboxyl-terminal domain of RNA Pol II, resulting in stimulation of transcription. Furthermore, the complexes containing CDK9 induce the differentiation in distinct tissue. The CDK9/cyclin T1 complex is expressed at higher level in more differentiated primary neuroectodermal and neuroblastoma tumors, showing a correlation between the kinase expression and tumor differentiation grade. This may have clinical and therapeutical implications for these tumor types. Among the CDK inhibitors two have shown to be effective against CDK9: Roscovitine and Flavopiridol. These two inhibitors prevented the replication of human immunodeficiency virus (HIV) type 1 by blocking Tat transactivation of the HIV type 1 promoter. These compounds inhibit CDKs by binding to the catalytic domain in place of ATP, preventing transfer of a phosphate group to the substrate. More sensitive therapeutic agents of CDK9 can be designed, and structural studies can add information in the understanding of this kinase. The major features related to CDK9 inhibition will be reviewed in this article.
Assuntos
Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/química , Sequência de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Quinase 9 Dependente de Ciclina/fisiologia , Flavonas/química , Flavonas/farmacologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Purinas/química , Purinas/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de SequênciaRESUMO
Cyclin-dependent kinase 11 is a relatively neglected member of the transcriptional CDKs subfamily, despite possibly being the most versatile CDK in this group. Different CDK11 variants are known to play essential roles in major cellular processes as mRNA transcription (CDK11p110), mitosis (CDK11p58), and apoptosis (CDK11p46 and CDK11p60). Each CDK11 species targets a particular set of substrates related to its functional background, but all isoforms originate from the CDC2L gene complex in human chromosome 1p36.2. CDK11p110 is synthesized through regular cap-dependent translation of CDK11 mRNA, whereas CDK11p58 translation is initiated through an IRES, and occurs only at G2 and M phases. CDK11p46 and CDK11p60, in turn, are the products of caspase cleavage of the larger isoforms during apoptosis. L-type cyclins are the main partners of CDK11, although CDK11p58 species interacts specifically with cyclin D3. The link between CDK11 dysfunction and cancer has been known for a long time, and critical roles in the proliferation of different cancer cell lines have been assigned to CDK11. This review summarizes more than 25 years of studies that unraveled CDK11 genetic and functional aspects.
Assuntos
Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Humanos , Neoplasias/patologiaRESUMO
An understanding of isoniazid (INH) drug resistance mechanism in Mycobacterium tuberculosis should provide significant insight for the development of newer anti-tubercular agents able to control INH-resistant tuberculosis (TB). The inhA-encoded 2-trans enoyl-acyl carrier protein reductase enzyme (InhA) has been shown through biochemical and genetic studies to be the primary target for INH. In agreement with these results, mutations in the inhA structural gene have been found in INH-resistant clinical isolates of M.tuberculosis, the causative agent of TB. In addition, the InhA mutants were shown to have higher dissociation constant values for NADH and lower values for the apparent first-order rate constant for INH inactivation as compared to wild-type InhA. Here, in trying to identify structural changes between wild-type and INH-resistant InhA enzymes, we have solved the crystal structures of wild-type and of S94A, I47T and I21V InhA proteins in complex with NADH to resolutions of, respectively, 2.3A, 2.2A, 2.0 A, and 1.9A. The more prominent structural differences are located in, and appear to indirectly affect, the dinucleotide binding loop structure. Moreover, studies on pre-steady-state kinetics of NADH binding have been carried out. The results showed that the limiting rate constant values for NADH dissociation from the InhA-NADH binary complexes (k(off)) were eleven, five, and tenfold higher for, respectively, I21V, I47T, and S94A INH-resistant mutants of InhA as compared to INH-sensitive wild-type InhA. Accordingly, these results are proposed to be able to account for the reduction in affinity for NADH for the INH-resistant InhA enzymes.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/química , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/química , Isoniazida/farmacologia , Mycobacterium tuberculosis/enzimologia , NAD/química , Oxirredutases/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/genética , Cinética , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Oxirredutases/genética , Ligação ProteicaRESUMO
CDK9 is a prominent member of the transcriptional CDKs subfamily, a group of kinases whose function is to control the primary steps of mRNA synthesis and processing by eukaryotic RNA polymerase II. As a cyclin-dependent kinase, CDK9 activation in vivo depends upon its association with T-type cyclins to assemble the positive transcription elongation factor (P-TEFb). Although CDK9/P-TEFb phosphorylates the C-terminal domain of RNAP II in the same positions targeted by CDK7 (TFIIH) and CDK8 (Mediator), the former does not participate in the transcription initiation, but rather plays a unique role by driving the polymerase to productive elongation. In addition to RNAP II CTD, the negative transcription elongation factors DSIF and NELF also represent major CDK9 substrates, whose phosphorylation is required to overcome the proximal pause of the polymerase. CDK9 is recruited to specific genes through proteins that interact with both P-TEFb and distinct elements in DNA, RNA or chromatin, where it modulates the activity of individual RNAP II transcription complexes. The regulation of CDK9 function is an intricate network that includes post-translational modifications (phosphorylation/dephosphorylation and acetylation/deacetylation of key residues) as well as the association of P-TEFb with various proteins that can stimulate or inhibit its kinase activity. Several cases of CDK9 deregulation have been linked to important human diseases, including various types of cancer and also AIDS (due to its essential role in HIV replication). Not only HIV, but also many other human viruses have been shown to depend strongly on CDK9 activity to be transcribed within host cells. This review summarizes the main advances made on CDK9/P-TEFb field in more than 20 years, introducing the structural, functional and genetic aspects that have been elucidated ever since.
Assuntos
Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Animais , Quinase 9 Dependente de Ciclina/química , Regulação da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/química , Ligação Proteica , Transcrição Gênica , Vírus/genéticaRESUMO
Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure- based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/química , Sítios de Ligação/efeitos dos fármacos , Simulação por Computador , Cristalização/métodos , Cristalografia por Raios X/métodos , Ligantes , Fosfatos/química , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína , Nucleosídeos de Purina , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Genome sequencing efforts are providing us with complete genetic blueprints for hundreds of organisms. We are now faced with assigning, understanding, and modifying the functions of proteins encoded by these genomes. DBMODELING is a relational database of annotated comparative protein structure models and their metabolic pathway characterization, when identified. This procedure was applied to complete genomes such as Mycobacterium tuberculosis and Xylella fastidiosa. The main interest in the study of metabolic pathways is that some of these pathways are not present in humans, which makes them selective targets for drug design, decreasing the impact of drugs in humans. In the database, there are currently 1,116 proteins from two genomes. It can be accessed by any researcher at http://www.biocristalografia.df.ibilce.unesp.br/tools/. This project confirms that homology modeling is a useful tool in structural bioinformatics and that it can be very valuable in annotating genome sequence information, contributing to structural and functional genomics, and analyzing protein-ligand docking.
Assuntos
Bases de Dados Factuais/estatística & dados numéricos , Bases de Dados de Proteínas/estatística & dados numéricos , Mycobacterium tuberculosis/genética , Proteínas/química , Xylella/genética , Animais , Sistemas de Gerenciamento de Base de Dados/instrumentação , Bases de Dados Genéticas/estatística & dados numéricos , Desenho de Fármacos , Genoma/genética , Genoma Bacteriano , Humanos , Imageamento Tridimensional/métodos , Armazenamento e Recuperação da Informação/métodos , Internet/instrumentação , Modelos Moleculares , Proteínas/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Homologia Estrutural de ProteínaRESUMO
The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the alpha-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.
Assuntos
Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Triptofano Sintase/antagonistas & inibidores , Triptofano Sintase/química , Sítios de Ligação , Simulação por Computador , Desenho de Fármacos , Fagaceae/genética , Ligação de Hidrogênio , Ligantes , Estrutura Molecular , Proteínas de Plantas/química , Proteínas de Plantas/classificação , Ligação Proteica/fisiologia , Conformação Proteica , Rosaceae/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8%(w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 A resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.
Assuntos
Fabaceae/química , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Sementes/química , Sequência de Aminoácidos , Animais , Cromatografia de Afinidade , Cristalização/métodos , Cristalografia por Raios X , Hemaglutinação , Manose/química , Dados de Sequência Molecular , Lectinas de Plantas/farmacologia , CoelhosRESUMO
A chitin-binding protein named PPL-2 was purified from Parkia platycephala seeds and crystallized. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 A, and grew over several days at 293 K using the hanging-drop method. Using synchrotron radiation, a complete structural data set was collected to 1.73 A resolution. The preliminary crystal structure of PPL-2, determined by molecular replacement, presents a correlation coefficient of 0.558 and an R factor of 0.439. Crystallographic refinement is in progress.
Assuntos
Quitina/metabolismo , Fabaceae/química , Proteínas de Plantas/química , Sementes/química , Sequência de Aminoácidos , Cristalização , Dados de Sequência Molecular , Ligação Proteica , Alinhamento de Sequência , Difração de Raios XRESUMO
A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.
Assuntos
Canavalia/química , Lectinas de Plantas/química , Sementes/química , Cristalização , Lectinas de Plantas/isolamento & purificação , Difração de Raios XRESUMO
Crystal structure of hemoglobin isolated from the Brazilian maned wolf (Chrysocyon brachyurus) was determined using standard molecular replacement technique and refined using maximum-likelihood and simulated annealing protocols to 1.87A resolution. Structural and functional comparisons between hemoglobins from the Chrysocyon brachyurus and Homo sapiens are discussed, in order to provide further insights in the comparative biochemistry of vertebrate hemoglobins.
Assuntos
Hemoglobinas/química , Lobos , Animais , Brasil , Cristalização , Hemoglobinas/isolamento & purificação , Histidina , Ferro , Oxiemoglobinas/química , Conformação Proteica , Síncrotrons , Difração de Raios XRESUMO
The flavoprotein old yellow enzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that uses NAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanism of some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In this work, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27Å showed a classical (α/ß)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein flexibility. Moreover, the binding mode of ß-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.
Assuntos
NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismo , Trypanosoma cruzi/enzimologia , Cristalografia por Raios X , Modelos Moleculares , NADPH Desidrogenase/genética , Conformação Proteica , Especificidade por Substrato , TermodinâmicaRESUMO
The complex CDK3-cyclin is involved in the control of the progression of G0. While the mechanisms governing early and late G1 progression are well understood, very little is known about the G0-G1 transition. Human CDK3 is closely related to cyclin-dependent kinase 2 (CDK2). Since there is no crystallographic structure of human CDK3, this work describes for the first time a molecular model of human CDK3 complexed with several inhibitors. Comparison of the binary complexes with different inhibitors strongly indicates that those inhibitors should inhibit CDK3 as well as CDK2.
Assuntos
Quinases Ciclina-Dependentes/química , Inibidores de Proteínas Quinases/química , Sequência de Aminoácidos , Quinase 3 Dependente de Ciclina , Quinases Ciclina-Dependentes/antagonistas & inibidores , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Inibidores de Proteínas Quinases/farmacologia , Homologia de Sequência de AminoácidosRESUMO
Protein crystallography is the main technique used to obtain three-dimensional information for binary complexes involving protein and drugs. Once a protein target has its three-dimensional structure elucidated, the next natural step is the solving of the structure complexed either with its natural substrate, or any ligand or even an inhibitor. Such information is of pivotal importance to understand the structural basis for inhibition of an enzyme. The relevant features, for application of protein crystallography to drug discovery, are discussed in this review.