RESUMO
The anticancer agent indisulam inhibits cell proliferation by causing degradation of RBM39, an essential mRNA splicing factor. Indisulam promotes an interaction between RBM39 and the DCAF15 E3 ligase substrate receptor, leading to RBM39 ubiquitination and proteasome-mediated degradation. To delineate the precise mechanism by which indisulam mediates the DCAF15-RBM39 interaction, we solved the DCAF15-DDB1-DDA1-indisulam-RBM39(RRM2) complex structure to a resolution of 2.3 Å. DCAF15 has a distinct topology that embraces the RBM39(RRM2) domain largely via non-polar interactions, and indisulam binds between DCAF15 and RBM39(RRM2), coordinating additional interactions between the two proteins. Studies with RBM39 point mutants and indisulam analogs validated the structural model and defined the RBM39 α-helical degron motif. The degron is found only in RBM23 and RBM39, and only these proteins were detectably downregulated in indisulam-treated HCT116 cells. This work further explains how indisulam induces RBM39 degradation and defines the challenge of harnessing DCAF15 to degrade additional targets.
Assuntos
Antineoplásicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Ligação a RNA/química , Sulfonamidas/farmacologia , Motivos de Aminoácidos , Calorimetria , Clonagem Molecular , Fluorometria , Células HCT116 , Células HEK293 , Humanos , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinética , Proteínas Nucleares/metabolismo , Peptídeos/química , Mutação Puntual , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteoma , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Proteínas de Transporte/metabolismo , Limoninas/farmacologia , Proteólise/efeitos dos fármacos , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Limoninas/química , Limoninas/isolamento & purificação , Ubiquitina-Proteína LigasesRESUMO
Glycogen synthase kinase-3 plays an essential role in multiple biochemical pathways in the cell, particularly in regards to energy regulation. As such, Glycogen synthase kinase-3 is an attractive target for pharmacological intervention in a variety of disease states, particularly non-insulin dependent diabetes mellitus. However, due to homology with other crucial kinases, such as the cyclin-dependent protein kinase CDC2, developing compounds that are both potent and selective is challenging. A novel series of derivatives of 5-nitro-N2-(2-(pyridine-2ylamino)ethyl)pyridine-2,6-diamine were synthesized and have been shown to potently inhibit glycogen synthase kinase-3 (GSK3). Potency in the low nanomolar range was obtained along with remarkable selectivity. The compounds activate glycogen synthase in insulin receptor-expressing CHO-IR cells and in primary rat hepatocytes, and have acceptable pharmacokinetics and pharmacodynamics to allow for oral dosing. The X-ray co-crystal structure of human GSK3-ß in complex with compound 2 is reported and provides insights into the structural determinants of the series responsible for its potency and selectivity.
Assuntos
Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Piridinas/química , Animais , Sítios de Ligação , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Quinase 3 da Glicogênio Sintase/metabolismo , Meia-Vida , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Estrutura Terciária de Proteína , Piridinas/metabolismo , Piridinas/farmacocinética , Ratos , Relação Estrutura-AtividadeRESUMO
Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2. The x-ray co-structures of the FluB PB2(cap) with bound cap analogs m(7)GTP and GTP reveal an inverted GTP binding mode that is distinct from the cognate m(7)GTP binding mode shared between FluA and FluB PB2. These results delineate the commonalities and differences in the cap-binding site between FluA and FluB PB2 and will aid structure-guided drug design efforts to identify dual inhibitors of both FluA and FluB PB2.
Assuntos
Vírus da Influenza B/enzimologia , Subunidades Proteicas/metabolismo , Capuzes de RNA/metabolismo , Proteínas Virais/metabolismo , Calorimetria , Cristalografia por Raios X , Fluorometria , Vírus da Influenza A/enzimologia , Modelos Moleculares , Maleabilidade , Subunidades Proteicas/química , Análogos de Capuz de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Soluções , Proteínas Virais/químicaRESUMO
Structure-based drug design is an integral part of modern day drug discovery and requires detailed structural characterization of protein-ligand interactions, which is most commonly performed by X-ray crystallography. However, the success rate of generating these costructures is often variable, in particular when working with dynamic proteins or weakly binding ligands. As a result, structural information is not routinely obtained in these scenarios, and ligand optimization is challenging or not pursued at all, representing a substantial limitation in chemical scaffolds and diversity. To overcome this impediment, we have developed a robust NMR restraint guided docking protocol to generate high-quality models of protein-ligand complexes. By combining the use of highly methyl-labeled protein with experimentally determined intermolecular distances, a comprehensive set of protein-ligand distances is generated which then drives the docking process and enables the determination of the correct ligand conformation in the bound state. For the first time, the utility and performance of such a method is fully demonstrated by employing the generated models for the successful, prospective optimization of crystallographically intractable fragment hits into more potent binders.
Assuntos
Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Proteínas/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Proteínas/metabolismoRESUMO
In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity. Structural studies demonstrate specific binding of the peptide to the pore of pentameric VP1. Cell-based assays demonstrate nanomolar inhibition (EC50) of BKV infection and suggest that the peptide acts early in the viral entry pathway. Homologous peptide exhibits similar binding to JC polyomavirus VP1 and inhibits infection with similar potency to BKV in a model cell line. Lastly, these studies validate targeting the VP1 pore as a novel strategy for the development of anti-polyomavirus agents.
Assuntos
Antivirais/metabolismo , Vírus BK , Proteínas do Capsídeo/metabolismo , Vírus JC/efeitos dos fármacos , Peptídeos/metabolismo , Antivirais/química , Antivirais/farmacologia , Vírus BK/efeitos dos fármacos , Vírus BK/genética , Vírus BK/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Células Cultivadas , Células HEK293 , Humanos , Peptídeos/química , Peptídeos/genética , Ligação ProteicaRESUMO
Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11-13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins. VX-787 is an inhibitor of the influenza A virus pre-mRNA cap-binding protein PB2. This clinical stage compound was shown to bind the minimal cap-binding domain of PB2 to inhibit the cap-snatching machinery. However, the binding of this molecule in the context of an extended form of the PB2 subunit has remained elusive. Here we generated a collection of PB2 truncations to identify a PB2 protein representative of its structure in the viral heterotrimeric protein. We present the crystal structure of VX-787 bound to a PB2 construct that recapitulates VX-787's biological antiviral activity in vitro. This co-structure reveals more extensive interactions than previously identified and provides insight into the observed resistance profile, affinity, binding kinetics, and conformational rearrangements induced by VX-787.
Assuntos
Antivirais/química , Vírus da Influenza A/enzimologia , Subunidades Proteicas/química , RNA Polimerase Dependente de RNA/química , Antivirais/farmacologia , Sítios de Ligação , Humanos , Vírus da Influenza A/efeitos dos fármacos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Subunidades Proteicas/antagonistas & inibidores , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
PRC2 is a multisubunit methyltransferase involved in epigenetic regulation of early embryonic development and cell growth. The catalytic subunit EZH2 methylates primarily lysine 27 of histone H3, leading to chromatin compaction and repression of tumor suppressor genes. Inhibiting this activity by small molecules targeting EZH2 was shown to result in antitumor efficacy. Here, we describe the optimization of a chemical series representing a new class of PRC2 inhibitors which acts allosterically via the trimethyllysine pocket of the noncatalytic EED subunit. Deconstruction of a larger and complex screening hit to a simple fragment-sized molecule followed by structure-guided regrowth and careful property modulation were employed to yield compounds which achieve submicromolar inhibition in functional assays and cellular activity. The resulting molecules can serve as a simplified entry point for lead optimization and can be utilized to study this new mechanism of PRC2 inhibition and the associated biology in detail.
Assuntos
Inibidores Enzimáticos/química , Epigênese Genética , Metiltransferases/antagonistas & inibidores , Complexo Repressor Polycomb 2/química , Regulação Alostérica , Células CACO-2 , Cromatografia Líquida , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Espectrometria de Massas , Estrutura Molecular , Espectroscopia de Prótons por Ressonância Magnética , Relação Estrutura-AtividadeRESUMO
In an effort to identify new antidiabetic agents, we have discovered a novel family of (5-imidazol-2-yl-4-phenylpyrimidin-2-yl)[2-(2-pyridylamino)ethyl]amine analogues which are inhibitors of human glycogen synthase kinase 3 (GSK3). We developed efficient synthetic routes to explore a wide variety of substitution patterns and convergently access a diverse array of analogues. Compound 1 (CHIR-911, CT-99021, or CHIR-73911) emerged from an exploration of heterocycles at the C-5 position, phenyl groups at C-4, and a variety of differently substituted linker and aminopyridine moieties attached at the C-2 position. These compounds exhibited GSK3 IC50s in the low nanomolar range and excellent selectivity. They activate glycogen synthase in insulin receptor-expressing CHO-IR cells and primary rat hepatocytes. Evaluation of lead compounds 1 and 2 (CHIR-611 or CT-98014) in rodent models of type 2 diabetes revealed that single oral doses lowered hyperglycemia within 60 min, enhanced insulin-stimulated glucose transport, and improved glucose disposal without increasing insulin levels.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinases da Glicogênio Sintase/antagonistas & inibidores , Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacologia , Pirimidinas/farmacologia , Animais , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetulus , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Humanos , Hipoglicemiantes/metabolismo , Espectrometria de Massas , Espectroscopia de Prótons por Ressonância Magnética , Pirimidinas/química , Pirimidinas/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.
Assuntos
Inibidores Enzimáticos/farmacologia , Simulação de Acoplamento Molecular , Complexo Repressor Polycomb 2/antagonistas & inibidores , Sulfonas/farmacologia , Triazóis/farmacologia , Animais , Sítios de Ligação , Descoberta de Drogas , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Camundongos , Complexo Repressor Polycomb 2/química , Complexo Repressor Polycomb 2/metabolismo , Relação Quantitativa Estrutura-Atividade , Sulfonas/química , Triazóis/químicaRESUMO
VC0702, a conserved hypothetical protein of unknown function from Vibrio cholerae, resides in a three-gene operon containing the MbaA gene that encodes for a GGDEF and EAL domain-containing protein which is involved in regulating formation of the extracellular matrix of biofilms in Vibrio cholerae. The VC0702 crystal structure has been determined at 2.0 A and refined to Rwork = 22.8% and Rfree = 26.3%. VC0702 crystallized in an orthorhombic crystal lattice in the C222(1) space group with dimensions of a = 66.61 A, b = 88.118 A, and c = 118.35 A with a homodimer in the asymmetric unit. VC0702, which forms a mixed alpha + beta three-layered alphabetaalpha sandwich, belongs to the Pfam DUF84 and COG1986 families of proteins. Sequence conservation within the DUF84 and COG1986 families was used to identify a conserved patch of surface residues that define a cleft and potential substrate-binding site in VC0702. The three-dimensional structure of VC0702 is similar to that of Mj0226 from Methanococcus janeschii, which has been identified as a novel NTPase that binds NTP in a deep cleft similarly located to the conserved patch of surface residues that define an analogous cleft in VC0702. Collectively, the data suggest that VC0702 may have a biochemical function that involves NTP binding and phosphatase activity of some kind, and is likely involved in regulation of the signaling pathway that controls biofilm formation and maintenance in Vibrio cholerae.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vibrio cholerae/química , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Purine-binding proteins are of critical importance to all living organisms. Approximately 13% of the human genome is devoted to coding for purine-binding proteins. Given their importance, purine-binding proteins are attractive targets for chemotherapeutic intervention against a variety of disease states, particularly cancer. Modern computational and biophysical techniques, combined together in a structure-based drug design approach, aid immensely in the discovery of inhibitors of these targets. This review covers the process of modern structure-based drug design and gives examples of its use in discovery and development of drugs that target purine-binding proteins. The targets reviewed are human purine nucleoside phosphorylase, human epidermal growth factor receptor kinase, and human kinesin spindle protein.
Assuntos
Antineoplásicos , Desenho de Fármacos , Neoplasias/tratamento farmacológico , Proteoma , Nucleotídeos de Purina , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Neoplasias/metabolismo , Proteoma/genética , Proteoma/metabolismo , Nucleotídeos de Purina/genética , Nucleotídeos de Purina/metabolismo , Relação Estrutura-AtividadeRESUMO
Originally identified as a modulator of glycogen metabolism, glycogen synthase kinase-3 (GSK3) is now understood to play an important regulatory role in a variety of pathways including initiation of protein synthesis, cell proliferation, cell differentiation, apoptosis, and is essential for embryonic development as a component of the Wnt signaling cascade. GSK3 can be considered as a target for both metabolic and neurological disorders. GSK3's association with neuronal apoptosis and hyper-phosphorylation of tau make this kinase an attractive therapeutic target for neurodegenerative conditions such as head trauma, stroke and Alzheimer's disease. While noting GSK3's many associated functions, this review will focus on GSK3 as a central negative regulator in the insulin signaling pathway, its role in insulin resistance, and the utility of GSK3 inhibitors for intervention and control of metabolic diseases including type 2 diabetes. Recent crystal structures, including the active (phosphorylated Tyr-216) form of GSK3beta, provide a wealth of structural information and greater understanding of GSK3's unique regulation and substrate specificity. Many potent and selective small molecule inhibitors of GSK3 have now been identified, and used in vitro to modulate glycogen metabolism and gene transcription, increase glycogen synthase activity and enhance insulin-stimulated glucose transport. The pharmacology of potent and selective GSK3 inhibitors (CT 99021 and CT 20026) is described in a number of in vitro and in vivo models following acute or chronic exposure. The efficacy of clinical candidates in diabetic primates and the implications for clinical development are discussed. The profile of activity is consistent with a unique form of insulin sensitization which is well suited for indications such as metabolic syndrome X and type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Inibidores Enzimáticos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hipoglicemiantes , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Modelos Moleculares , Estrutura Molecular , Especificidade por SubstratoRESUMO
Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.
Assuntos
Autofagia/fisiologia , Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Ferritinas/metabolismo , Homeostase/fisiologia , Ferro/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Animais , Autofagia/efeitos dos fármacos , Células Cultivadas , Humanos , Lisossomos/metabolismo , Camundongos , Fagossomos/metabolismo , Ligação ProteicaRESUMO
Purines are critical cofactors in the enzymatic reactions that create and maintain living organisms. In humans, there are approximately 3,266 proteins that utilize purine cofactors and these proteins constitute the so-called purinome. The human purinome encompasses a wide-ranging functional repertoire and many of these proteins are attractive drug targets. For example, it is estimated that 30% of modern drug discovery projects target protein kinases and that modulators of small G-proteins comprise more than 50% of currently marketed drugs. Given the importance of purine-binding proteins to drug discovery, the following review will discuss the forces that mediate protein:purine recognition, the factors that determine druggability of a protein target, and the process of structure-based drug design. A review of purine recognition in representatives of the various purine-binding protein families, as well as the challenges faced in targeting members of the purinome in drug discovery campaigns will also be given.
Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Purinas/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Humanos , Cinética , Ligantes , Modelos Moleculares , Biologia Molecular/métodos , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/genética , Ligação Proteica , Conformação Proteica , Proteínas Quinases/química , Proteínas Quinases/efeitos dos fármacos , Purinas/metabolismo , Design de Software , Termodinâmica , Proteínas ras/química , Proteínas ras/efeitos dos fármacos , Proteínas ras/genéticaRESUMO
CHK-1 is one of the key enzymes regulating checkpoints in cellular growth cycles. Novel 4-(amino-alkylamino)-3-benzimidazole-quinolinones were prepared and assayed for their ability to inhibit CHK-1. These compounds are potent cell permeable CHK-1 inhibitors and showed synergistic effect with a DNA-damaging agent, camptothecin.
Assuntos
Benzimidazóis/química , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Quinolonas/química , Quinolonas/farmacologia , Alquilação , Aminação , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Cristalografia por Raios X , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Quinolonas/síntese química , Relação Estrutura-AtividadeRESUMO
Isopentenyl pyrophosphate (IPP) is a universal building block for the ubiquitous isoprenoids that are essential to all organisms. The enzymes of the non-mevalonate pathway for IPP synthesis, which is unique to many pathogenic bacteria, have recently been explored as targets for antibiotic development. Several crystal structures of 2C-methyl-D-erythritol-2,4-cyclophosphate (MECDP) synthase, the fifth of seven enzymes involved in the non-mevalonate pathway for synthesis of IPP, have been reported; however, the composition of metal ions in the active site and the presence of a hydrophobic cavity along the non-crystallographic threefold symmetry axis has varied between the reported structures. Here, the structure of MEDCP from Shewanella oneidensis MR1 (SO3437) was determined to 1.6 A resolution in the absence of substrate. The presence of a zinc ion in the active-site cleft, tetrahedrally coordinated by two histidine side chains, an aspartic acid side chain and an ambiguous fourth ligand, was confirmed by zinc anomalous diffraction. Based on analysis of anomalous diffraction data and typical metal-to-ligand bond lengths, it was concluded that an octahedral sodium ion was 3.94 A from the zinc ion. A hydrophobic cavity was observed along the threefold non-crystallographic symmetry axis, filled by a well defined non-protein electron density that could be modeled as farnesyl pyrophosphate (FPP), a downstream product of IPP, suggesting a possible feedback mechanism for enzyme regulation. The high-resolution data clarified the FPP-binding mode compared with previously reported structures. Multiple sequence alignment indicated that the residues critical to the formation of the hydrophobic cavity and for coordinating the pyrophosphate group of FPP are present in the majority of MEDCP synthase enzymes, supporting the idea of a specialized biological function related to FPP binding in a subfamily of MEDCP synthase homologs.
Assuntos
Aldose-Cetose Isomerases/química , Complexos Multienzimáticos/química , Oxirredutases/química , Fosfatos de Poli-Isoprenil/química , Shewanella/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Elétrons , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , SesquiterpenosRESUMO
CylR2 is one of two regulatory proteins associated with the quorum-sensing-dependent synthesis of cytolysin in the common pathogen Enterococcus faecalis. The protein was expressed with a C-terminal six-histidine tag and purified to homogeneity with a cobalt-affinity column followed by size-exclusion chromatography. Both native and SeMet proteins were produced and crystallized. Complete X-ray diffraction data sets were collected from a native crystal, which diffracted to 2.3 angstroms resolution, and a SeMet crystal, which diffracted to 2.1 angstroms. The crystals were tetragonal, belonging to space group P4(1) or P4(3), with unit-cell parameters a = b = 66.2, c = 40.9 angstroms, alpha = beta = gamma = 90 degrees. Based on the calculated Matthews coefficient of 2.6 angstroms3 Da(-1) as well as analysis of anomalous difference Patterson maps, the asymmetric unit most likely contains two molecules of CylR2.