RESUMO
The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.
Assuntos
Burkholderiales , Burkholderiales/enzimologia , Burkholderiales/genética , Burkholderiales/metabolismo , Ácidos Ftálicos/metabolismo , Ácidos Ftálicos/química , Hidrolases/metabolismo , Hidrolases/genética , Hidrolases/química , Solubilidade , Polietilenotereftalatos/metabolismo , Polietilenotereftalatos/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Engenharia de Proteínas/métodos , Dobramento de Proteína , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Modelos MolecularesRESUMO
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.
Assuntos
Amidas , Proteínas Periplásmicas de Ligação , Amidas/metabolismo , Amidas/química , Cristalografia por Raios X , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Periplásmicas de Ligação/química , Proteínas Periplásmicas de Ligação/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Aminoácidos/metabolismo , Mesorhizobium/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Modelos Moleculares , Amidoidrolases/metabolismo , Amidoidrolases/química , Cálcio/metabolismo , Ligação ProteicaRESUMO
Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.
Assuntos
Leucina , Leucina/química , Escherichia coli/metabolismo , Conformação Proteica , Modelos Moleculares , Cristalografia por Raios X , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismoRESUMO
Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61â Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.
Assuntos
Elementos da Série dos Lantanídeos , Metaloproteínas , Hidrolases de Triester Fosfórico , Hidrolases de Triester Fosfórico/química , Hidrolases de Triester Fosfórico/metabolismo , Domínio Catalítico , Gadolínio , Európio , CátionsRESUMO
Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.
Assuntos
Insulinas , PPAR gama , Proteínas Correpressoras/metabolismo , Agonismo Inverso de Drogas , Ligantes , PPAR gama/metabolismo , Conformação ProteicaRESUMO
The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene-reductase activity, the old yellow enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F420. Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction.
Assuntos
Mycobacterium smegmatis , Oxirredutases , Oxirredutases/metabolismo , Mycobacterium smegmatis/metabolismo , Oxirredução , NADPH Desidrogenase/química , NADPH Desidrogenase/metabolismoRESUMO
Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.
RESUMO
Cyanopyridylalanines are non-canonical amino acids that react with aminothiol compounds under physiological conditions in a biocompatible manner without requiring added catalyst. Here we present newly developed aminoacyl-tRNA synthetases for genetic encoding of meta- and para-cyanopyridylalanine to enable the site-specific attachment of a wide range of different functionalities. The outstanding utility of the cyanopyridine moiety is demonstrated by examples of i) post-translational functionalization of proteins, ii)â in-cell macrocyclization of peptides and proteins, and iii)â protein stapling. The biocompatible nature of the protein ligation chemistry enabled by the cyanopyridylalanine amino acid opens a new path to specific in vivo protein modifications in complex biological environments.
Assuntos
Aminoacil-tRNA Sintetases , Nitrilas , Aminas , Aminoácidos/química , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Proteínas/química , Compostos de SulfidrilaRESUMO
Fluorine atoms are known to display scalar 19F-19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space 19F-19F (TSJFF) couplings even if the 19F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF3 groups, O-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that 19F-19F TOCSY experiments are sufficiently sensitive to detect TSJFF couplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative J evolution experiment enables the measurement of TSJFF coupling constants that are up to five times smaller than the 19F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of N6-(trifluoroacetyl)-l-lysine (TFA-Lys) and 19F-19F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding, 19F-19F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of 1H NMR resonances.
Assuntos
Aminoácidos/análise , Peptidilprolil Isomerase/química , Flúor , Halogenação , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
A promising approach for treating type 2 diabetes mellitus (T2DM) is to target the Peroxisome Proliferator-Activated Receptor γ (PPARγ) transcription factor, which regulates the expression of proteins critical for T2DM. Mechanisms involved in PPARγ signaling are poorly understood, yet globally increasing T2DM prevalence demands improvements in drug design. Synthetic, nonactivating PPARγ ligands can abolish the phosphorylation of PPARγ at Ser273, a posttranslational modification correlated with obesity and insulin resistance. It is not understood how these ligands prevent phosphorylation, and the lack of experimental mechanistic information can be attributed to previous ambiguity in the field as well as to limitations in experimental approaches; in silico modeling currently provides the only insight into how ligands block Ser273 phosphorylation. The future availability of experimental evidence is critical for clarifying the mechanism by which ligands prevent phosphorylation and should be the priority of future T2DM-focused research. Following this, the properties of ligands that enable them to block phosphorylation can be improved upon to generate ligands tailored for blocking phosphorylation and therefore restoring insulin sensitivity. This would represent a significant step forward for treating T2DM. This review summarizes current knowledge of the roles of PPARγ in T2DM as well as the effects of synthetic ligands on the modulation of these roles. We hypothesize potential factors that contribute to the reduction in recent developments and summarize what has currently been done to shed light on this critical field of research.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , PPAR gama/antagonistas & inibidores , Humanos , Resistência à Insulina , Ligantes , PPAR gama/química , PPAR gama/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Serina/metabolismoRESUMO
Obtaining high-throughput structural characterization of small molecule drug candidates bound to their protein target has been hampered by the challenges of cocrystallizing protein-ligand complexes. These challenges include poor ligand solubility, excess ligand molecules disrupting the homogeneity of the sample to be crystallized, and inefficiency in preparing individual complexes and crystal screens for each drug candidate. Crystallizing apo protein followed by soaking with a solution containing the ligand of interest is a powerful tool for rapidly obtaining structural information of ligands. Here, we describe the process of purifying, crystallizing, and soaking PPARγ ligand binding domain as well as strategies for cocrystallizing ligands that are not amenable to soaking.
Assuntos
Cristalização/métodos , PPAR gama/química , PPAR gama/metabolismo , Escherichia coli/genética , Humanos , Ligantes , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Obesity and rheumatic disease are mechanistically linked via chronic inflammation. The orphan receptor TREM-1 (triggering receptor expressed on myeloid cells-1) is a potent amplifier of proinflammatory and noninfectious immune responses. Here, we show that the pan modulator SR1903 effectively blocks TREM-1 activation. SR1903 emerged from a chemical series of potent RORγ inverse agonists, although unlike close structural analogues, it has modest agonist activity on LXR and weak repressive activity (inverse agonism) of PPARγ, three receptors that play essential roles in inflammation and metabolism. The anti-inflammatory and antidiabetic efficacy of this unique modulator in collagen-induced arthritis and diet-induced obesity mouse models is demonstrated. Interestingly, in the context of obesity, SR1903 aided in the maintenance of the thymic homeostasis unlike selective RORγ inverse agonists. SR1903 was well-tolerated following chronic administration, and combined, these data suggest that it may represent a viable strategy for treatment of both metabolic and inflammatory disease. More importantly, the ability of SR1903 to block LPS signaling suggests the potential utility of this unique polypharmacological modulator for treatment of innate immune response disorders.
Assuntos
Compostos de Bifenilo/farmacologia , Inflamação/metabolismo , Piperazinas/farmacologia , Polifarmacologia , Propanóis/farmacologia , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Animais , Artrite Experimental/tratamento farmacológico , Compostos de Bifenilo/uso terapêutico , Dieta , Modelos Animais de Doenças , Agonismo Inverso de Drogas , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Obesidade/tratamento farmacológico , Obesidade/etiologia , PPAR gama/agonistas , PPAR gama/metabolismo , Piperazinas/uso terapêutico , Propanóis/uso terapêutico , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismoRESUMO
In search for effective multi-targeting drug ligands (MTDLs) to address low-grade inflammatory changes of metabolic disorders, we rationally designed some novel glitazones-like compounds. This was achieved by incorporating prominent pharmacophoric motifs from previously reported COX-2, 15-LOX and PPARγ ligands. Challenging our design with pre-synthetic docking experiments on PPARγ showed encouraging results. In vitro tests have identified 4 compounds as simultaneous partial PPARγ agonist, potent COX-2 antagonist (nanomolar IC50 values) and moderate 15-LOX inhibitor (micromolar IC50 values). We envisioned such outcome as a prototypical balanced modulation of the 3 inflammatory targets. In vitro glucose uptake assay defined six compounds as insulin-sensitive and the other two as insulin-independent glucose uptake enhancers. Also, they were able to induce PPARγ nuclear translocation in immunohistochemical analysis. Their anti-inflammatory potential has been translated to effective inhibition of monocyte to macrophage differentiation, suppression of LPS-induced inflammatory cytokine production in macrophages, as well as significant in vivo anti-inflammatory activity. Ligand co-crystallized PPARγ X-ray of one of MTDLs has identified new clues that could serve as structural basis for its partial agonism. Docking of the most active compounds into COX-2 and 15-LOX active sites, pinpointed favorable binding patterns, similar to those of the co-crystallized ligands. Finally, in silico assessment of pharmacokinetics, physicochemical properties, drug-likeness and ligand efficiency indices was performed. Hence, we anticipate that the prominent biological profile of such series will rationalize relevant anti-inflammatory drug development endeavors.
Assuntos
Anti-Inflamatórios/química , Desenho de Fármacos , Tiazolidinedionas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Araquidonato 15-Lipoxigenase/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Humanos , Ligantes , Simulação de Acoplamento Molecular , PPAR gama/agonistas , Ligação Proteica , Tiazolidinedionas/química , Tiazolidinedionas/uso terapêuticoRESUMO
Peroxisome proliferator activated receptor γ (PPARγ) is a nuclear receptor and target for antidiabetics that increase insulin sensitivity. Owing to the side effects of PPARγ full agonists, research has recently focused on non-activating ligands of PPARγ, which increase insulin sensitivity with decreased side effects. Here, we present the crystal structures of inverse agonist SR10171 and a chemically related antagonist SR11023 bound to the PPARγ ligand-binding domain, revealing an allosteric switch in the activation helix, helix 12 (H12), forming an antagonist conformation in the receptor. H12 interacts with the antagonists to become fixed in an alternative location. Native mass spectrometry indicates that this prevents contacts with coactivator peptides and allows binding of corepressor peptides. Antagonists of related nuclear receptors act to sterically prevent the active configuration of H12, whereas these antagonists of PPARγ alternatively trap H12 in an inactive configuration, which we have termed the tumble and trap mechanism.
RESUMO
Targeting peroxisome proliferator-activated receptor γ (PPARγ) by synthetic compounds has been shown to elicit insulin sensitising properties in type 2 diabetics. Treatment with a class of these compounds, the thiazolidinediones (TZDs), has shown adverse side effects such as weight gain, fluid retention, and congestive heart failure. This is due to their full agonist properties on the receptor, where a number of genes are upregulated beyond normal physiological levels. Lessened transactivation of PPARγ by partial agonists has proved beneficial in terms of reducing side effects, while still maintaining insulin sensitising properties. However, some partial agonists have been associated with unfavourable pharmacokinetic profiles due to their acidic moieties, often causing partitioning to the liver. Here we present SR1988, a new partial agonist with favourable non-acid chemical properties. We used a combination of X-ray crystallography and hydrogen/deuterium exchange (HDX) to elucidate the structural basis for reduced activation of PPARγ by SR1988. This structural analysis reveals a mechanism that decreases stabilisation of the AF2 coactivator binding surface by the ligand.
RESUMO
Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor central to fatty acid and glucose homeostasis. PPARγ is the molecular target for type 2 diabetes mellitus (T2DM) therapeutics TZDs (thiazolidinediones), full agonists of PPARγ with robust antidiabetic properties, which are confounded with significant side effects. Partial agonists of PPARγ, such as INT131 (1), have displayed similar insulin-sensitizing efficacy as TZDs, but lack many side effects. To probe the structure-activity relationship (SAR) of the scaffold 1, we synthesized 14 analogs of compound 1 which revealed compounds with higher transcriptional potency for PPARγ and identification of moieties of the scaffold 1 key to high transcriptional potency. The sulfonamide linker is critical to activity, substitutions at position 4 of the benzene ring A were associated with higher transcriptional activity, substitutions at position 2 aided in tighter packing and activity, and the ring type and size of ring A affected the degree of activity.