RESUMO
Branching enzymes (BEs) are essential in the biosynthesis of starch and glycogen and play critical roles in determining the fine structure of these polymers. The substrates of these BEs are long carbohydrate chains that interact with these enzymes via multiple binding sites on the enzyme's surface. By controlling the branched-chain length distribution, BEs can mediate the physiological properties of starch and glycogen moieties; however, the mechanism and structural determinants of this specificity remain mysterious. In this study, we identify a large dodecaose binding surface on rice BE I (BEI) that reaches from the outside of the active site to the active site of the enzyme. Mutagenesis activity assays confirm the importance of this binding site in enzyme catalysis, from which we conclude that it is likely the acceptor chain binding site. Comparison of the structures of BE from Cyanothece and BE1 from rice allowed us to model the location of the donor-binding site. We also identified two loops that likely interact with the donor chain and whose sequences diverge between plant BE1, which tends to transfer longer chains, and BEIIb, which transfers exclusively much shorter chains. When the sequences of these loops were swapped with the BEIIb sequence, rice BE1 also became a short-chain transferring enzyme, demonstrating the key role these loops play in specificity. Taken together, these results provide a more complete picture of the structure, selectivity, and activity of BEs.
Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Cyanothece , Oryza , Enzima Ramificadora de 1,4-alfa-Glucana/química , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Glicogênio , Oryza/enzimologia , Oryza/metabolismo , Amido/biossíntese , Relação Estrutura-AtividadeRESUMO
Domain-swapping is a mechanism for evolving new protein structure from extant scaffolds, and has been an efficient protein-engineering strategy for tailoring functional diversity. However, domain swapping can only be exploited if it can be controlled, especially in cases where various folds can coexist. Herein, we describe the structure of a domain-swapped trimer of the iLBP family member hCRBPII, and suggest a mechanism for domain-swapped trimerization. It is further shown that domain-swapped trimerization can be favored by strategic installation of a disulfide bond, thus demonstrating a strategy for fold control. We further show the domain-swapped trimer to be a useful protein design template by installing a high-affinity metal binding site through the introduction of a single mutation, taking advantage of its threefold symmetry. Together, these studies show how nature can promote oligomerization, stabilize a specific oligomer, and generate new function with minimal changes to the protein sequence.
Assuntos
Engenharia de Proteínas , Proteínas Celulares de Ligação ao Retinol/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica , Dobramento de ProteínaRESUMO
Protein conformational switches or allosteric proteins play a key role in the regulation of many essential biological pathways. Nonetheless, the implementation of protein conformational switches in protein design applications has proven challenging, with only a few known examples that are not derivatives of naturally occurring allosteric systems. We have discovered that the domain-swapped (DS) dimer of hCRBPII undergoes a large and robust conformational change upon retinal binding, making it a potentially powerful template for the design of protein conformational switches. Atomic resolution structures of the apo- and holo-forms illuminate a simple, mechanical movement involving sterically driven torsion angle flipping of two residues that drive the motion. We further demonstrate that the conformational "readout" can be altered by addition of cross-domain disulfide bonds, also visualized at atomic resolution. Finally, as a proof of principle, we have created an allosteric metal binding site in the DS dimer, where ligand binding results in a reversible 5-fold loss of metal binding affinity. The high resolution structure of the metal-bound variant illustrates a well-formed metal binding site at the interface of the two domains of the DS dimer and confirms the design strategy for allosteric regulation.
Assuntos
Engenharia de Proteínas/métodos , Proteínas Celulares de Ligação ao Retinol/química , Proteínas Celulares de Ligação ao Retinol/metabolismo , Regulação Alostérica , Sítios de Ligação , Dicroísmo Circular , Cristalografia por Raios X , Dissulfetos/química , Ligantes , Metais/metabolismo , Modelos Moleculares , Mutação , Domínios Proteicos , Multimerização Proteica , Proteínas Celulares de Ligação ao Retinol/genética , Treonina/genética , Tirosina/genética , Zinco/metabolismoRESUMO
Pancreatic cancer cells are characterized by deregulated metabolic programs that facilitate growth and resistance to oxidative stress. Among these programs, pancreatic cancers preferentially utilize a metabolic pathway through the enzyme aspartate aminotransferase 1 [also known as glutamate oxaloacetate transaminase 1 (GOT1)] to support cellular redox homeostasis. As such, small molecule inhibitors that target GOT1 could serve as starting points for the development of new therapies for pancreatic cancer. We ran a high-throughput screen for inhibitors of GOT1 and identified a small molecule, iGOT1-01, with in vitro GOT1 inhibitor activity. Application in pancreatic cancer cells revealed metabolic and growth inhibitory activity reflecting a promiscuous inhibitory profile. We then performed an in silico docking analysis to study inhibitor-GOT1 interactions with iGOT1-01 analogues that possess improved solubility and potency properties. These results suggested that the GOT1 inhibitor competed for binding to the pyridoxal 5-phosphate (PLP) cofactor site of GOT1. To analyze how the GOT1 inhibitor bound to GOT1, a series of GOT1 mutant enzymes that abolished PLP binding were generated. Application of the mutants in X-ray crystallography and thermal shift assays again suggested but were unable to formally conclude that the GOT1 inhibitor bound to the PLP site. Mutational studies revealed the relationship between PLP binding and the thermal stability of GOT1 while highlighting the essential nature of several residues for GOT1 catalytic activity. Insight into the mode of action of GOT1 inhibitors may provide leads to the development of drugs that target redox balance in pancreatic cancer.
Assuntos
Aspartato Aminotransferase Citoplasmática/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Inibidores Enzimáticos/farmacologia , Mutação , Neoplasias Pancreáticas/patologia , Aspartato Aminotransferase Citoplasmática/genética , Aspartato Aminotransferase Citoplasmática/metabolismo , Sítios de Ligação , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , Metabolômica , Modelos Moleculares , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Cellular retinol-binding proteins (CRBPs) I and II, which are members of the intracellular lipid-binding protein (iLBP) family, are retinoid chaperones that are responsible for the intracellular transport and delivery of both retinol and retinal. Although structures of retinol-bound CRBPI and CRBPII are known, no structure of a retinal-bound CRBP has been reported. In addition, the retinol-bound human CRBPII (hCRBPII) structure shows partial occupancy of a noncanonical conformation of retinol in the binding pocket. Here, the structure of retinal-bound hCRBPII and the structure of retinol-bound hCRBPII with retinol fully occupying the binding pocket are reported. It is further shown that the retinoid derivative seen in both the zebrafish CRBP and the hCRBPII structures is likely to be the product of flux-dependent and wavelength-dependent X-ray damage during data collection. The structures of retinoid-bound CRBPs are compared and contrasted, and rationales for the differences in binding affinities for retinal and retinol are provided.
Assuntos
Retinaldeído/metabolismo , Proteínas Celulares de Ligação ao Retinol/química , Proteínas Celulares de Ligação ao Retinol/metabolismo , Vitamina A/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Retinaldeído/química , Vitamina A/químicaRESUMO
Several novel and effective cysteine targeting (Cys) covalent drugs are in clinical use. However, the target area containing a druggable Cys residue is limited. Therefore, methods for creating covalent drugs that target different residues are being looked for; examples of such ligands include those that target the residues lysine (Lys) and tyrosine (Tyr). Though the histidine (His) side chain is more frequently found in protein binding locations and has higher desirable nucleophilicity, surprisingly limited research has been done to specifically target this residue, and there are not many examples of His-targeting ligands that have been rationally designed. In the current work, we created novel stapled peptides that are intended to target hMcl-1 His 252 covalently. We describe the in vitro (biochemical, NMR, and X-ray) and cellular design and characterization of such agents. Our findings further suggest that the use of electrophiles to specifically target His residues is warranted.
Assuntos
Histidina , Peptídeos , Histidina/química , Humanos , Peptídeos/química , Peptídeos/farmacologia , Conformação Proteica em alfa-Hélice , Cristalografia por Raios X , Modelos Moleculares , Desenho de Fármacos , LigantesRESUMO
Several investigational nitric oxide donors were originally created to correct vascular endothelial dysfunction in cardiovascular diseases. These 48 compounds contain an urea-like moiety attached to the well-known NO donors isosorbide 2- and 5-mononitrate. CR-0305 and CR-0202 were synthesized and found to be nontoxic in the cell lines HMEC-1, A549/hACE2 and VeroE6. CR-0305 induced vasodilation in human coronary arteries ex vivo. Since NO can also have antiviral properties, a study of drug-protein interactions with SARS-CoV-2 was undertaken using in silico modeling. CR-0305 experimentally outperformed the other compounds, including CR-0202, in binding the catalytic site of SARS-CoV-2 papain-like protease (PLpro). PLpro is a primary target for therapeutic inhibition of SARS-CoV-2 as it mediates viral replication and modulates host innate immune responses. CR-0305 is predicted to sit firmly in the PLpro catalytic pocket as confirmed by molecular dynamics simulations, wherein stability of binding to the catalytic site of PLpro induces a conformational change in the BL2 loop to a more closed conformation as observed previously with GRL0617. Surface plasmon resonance was performed with CR-0305 and CR-0202 to characterize binding affinity to purified SARS-CoV-2 PLpro protein. CR-0305 and CR-0202 also inhibited SARS-CoV-2 infection compared to vehicle as measured by virus N protein staining with a specific antibody in A549-ACE2 and VeroE6 cells at 20⯵M. CR-0305 is a coronary vasodilator that appears to bind to the catalytic site of the PLpro of SARS-CoV-2 while targeting delivery of antiviral NO to cells infected by SARS-CoV-2, suggesting multiple indications for future development.
Assuntos
COVID-19 , Peptídeo Hidrolases , Humanos , Papaína , SARS-CoV-2 , Doadores de Óxido Nítrico/farmacologia , Vasodilatadores , Antivirais/farmacologia , Inibidores de Proteases , Simulação de Acoplamento MolecularRESUMO
Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.
RESUMO
Significance: Protein disulfide isomerase (PDI) and endoplasmic reticulum oxidoreductase 1 (ERO1) are crucial for oxidative protein folding in the endoplasmic reticulum (ER). These enzymes are frequently overexpressed and secreted, and they contribute to the pathology of neurodegenerative, cardiovascular, and metabolic diseases. Recent Advances: Tissue-specific knockout mouse models and pharmacologic inhibitors have been developed to advance our understanding of the cell-specific functions of PDI and ERO1. In addition to their roles in protecting cells from the unfolded protein response and oxidative stress, recent studies have revealed that PDI and ERO1 also function outside of the cells. Critical Issues: Despite the well-known contributions of PDI and ERO1 to specific disease pathology, the detailed molecular and cellular mechanisms underlying these activities remain to be elucidated. Further, although PDI and ERO1 inhibitors have been identified, the results from previous studies require careful evaluation, as many of these agents are not selective and may have significant cytotoxicity. Future Directions: The functions of PDI and ERO1 in the ER have been extensively studied. Additional studies will be required to define their functions outside the ER.
Assuntos
Doenças Cardiovasculares/metabolismo , Glicoproteínas de Membrana/metabolismo , Doenças Metabólicas/metabolismo , Doenças Neurodegenerativas/metabolismo , Oxirredutases/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.
Assuntos
Desenho de Fármacos , Lisina/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Células A549 , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Simulação de Dinâmica Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30â mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Di-Inos/farmacologia , Inibidores Enzimáticos/farmacologia , Coração/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Antibacterianos/toxicidade , Proteínas de Bactérias/antagonistas & inibidores , Cardiotoxicidade , Di-Inos/síntese química , Di-Inos/farmacocinética , Di-Inos/toxicidade , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/toxicidade , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/farmacocinética , Ácidos Hidroxâmicos/toxicidade , Masculino , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pró-Fármacos/toxicidade , Pseudomonas aeruginosa/efeitos dos fármacos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Human Cellular Retinol Binding Protein II (hCRBPII), a member of the intracellular lipid-binding protein family, is a monomeric protein responsible for the intracellular transport of retinol and retinal. Herein we report that hCRBPII forms an extensive domain-swapped dimer during bacterial expression. The domain-swapped region encompasses almost half of the protein. The dimer represents a novel structural architecture with the mouths of the two binding cavities facing each other, producing a new binding cavity that spans the length of the protein complex. Although wild-type hCRBPII forms the dimer, the propensity for dimerization can be substantially increased via mutation at Tyr60. The monomeric form of the wild-type protein represents the thermodynamically more stable species, making the domain-swapped dimer a kinetically trapped entity. Hypothetically, the wild-type protein has evolved to minimize dimerization of the folding intermediate through a critical hydrogen bond (Tyr60-Glu72) that disfavors the dimeric form.