RÉSUMÉ
Loss-of-function mutations in the homotrimeric serine protease HTRA1 cause cerebral vasculopathy. Here, we establish independent approaches to achieve the functional correction of trimer assembly defects. Focusing on the prototypical R274Q mutation, we identify an HTRA1 variant that promotes trimer formation thus restoring enzymatic activity in vitro. Genetic experiments in Htra1R274Q mice further demonstrate that expression of this protein-based corrector in trans is sufficient to stabilize HtrA1-R274Q and restore the proteomic signature of the brain vasculature. An alternative approach employs supramolecular chemical ligands that shift the monomer-trimer equilibrium towards proteolytically active trimers. Moreover, we identify a peptidic ligand that activates HTRA1 monomers. Our findings open perspectives for tailored protein repair strategies.
Sujet(s)
High-temperature requirement A serine peptidase 1 , High-temperature requirement A serine peptidase 1/métabolisme , High-temperature requirement A serine peptidase 1/génétique , Animaux , Humains , Souris , Conformation des protéines , Multimérisation de protéines , Cellules HEK293 , Encéphale/métabolisme , Encéphale/anatomopathologie , Mutation , Mutation perte de fonctionRÉSUMÉ
SignificanceClassic serine proteases are synthesized as inactive precursors that are proteolytically processed, resulting in irreversible activation. We report an alternative and reversible mechanism of activation that is executed by an inactive protease. This mechanism involves a protein complex between the serine protease HTRA1 and the cysteine protease calpain 2. Surprisingly, activation is restricted as it improves the proteolysis of soluble tau protein but not the dissociation and degradation of its amyloid fibrils, a task that free HTRA1 is efficiently performing. These data exemplify a challenge for protein quality control proteases in the clearing of pathogenic fibrils and suggest a potential for unexpected side effects of chemical modulators targeting PDZ or other domains located at a distance to the active site.
Sujet(s)
Calpain , Serine endopeptidases , Amyloïde/métabolisme , Calpain/métabolisme , High-temperature requirement A serine peptidase 1/composition chimique , Protéolyse , Serine endopeptidases/métabolisme , Protéases à sérine/métabolismeRÉSUMÉ
DNA-scaffolded enzymes typically show altered kinetic properties; however, the mechanism behind this phenomenon is still poorly understood. We address this question using thrombin, a model of allosterically regulated serine proteases, encaged into DNA origami cavities with distinct structural and electrostatic features. We compare the hydrolysis of substrates that differ only in their net charge due to a terminal residue far from the cleavage site and presumably involved in the allosteric activation of thrombin. Our data show that the reaction rate is affected by DNA/substrate electrostatic interactions, proportionally to the degree of DNA/enzyme tethering. For substrates of opposite net charge, this leads to an inversion of the catalytic response of the DNA-scaffolded thrombin when compared to its freely diffusing counterpart. Hence, by altering the electrostatic environment nearby the encaged enzyme, DNA nanostructures interfere with charge-dependent mechanisms of enzyme-substrate recognition and may offer an alternative tool to regulate allosteric processes through spatial confinement.
RÉSUMÉ
Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.
Sujet(s)
Antiviraux/composition chimique , Antiviraux/pharmacologie , Composés pontés/pharmacologie , Organophosphates/pharmacologie , Protéines de l'enveloppe virale/effets des médicaments et des substances chimiques , Acid phosphatase/composition chimique , Acid phosphatase/métabolisme , Amyloïde/antagonistes et inhibiteurs , Agents antiVIH/composition chimique , Agents antiVIH/pharmacologie , Arginine/composition chimique , Betacoronavirus/effets des médicaments et des substances chimiques , Composés pontés/composition chimique , Membrane cellulaire/composition chimique , Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/virologie , Infections à VIH/traitement médicamenteux , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , Humains , Lipides/composition chimique , Lysine/composition chimique , Spectroscopie par résonance magnétique , Organophosphates/composition chimique , SARS-CoV-2 , Protéines sécrétoires des vésicules séminales/composition chimique , Protéines sécrétoires des vésicules séminales/métabolisme , Relation structure-activité , Protéines de l'enveloppe virale/métabolisme , Virus Zika/effets des médicaments et des substances chimiquesRÉSUMÉ
The interaction between the adapter protein 14-3-3σ and transcription factor p53 is important for preserving the tumor-suppressor functions of p53 in the cell. A phosphorylated motif within the C-terminal domain (CTD) of p53 is key for binding to the amphipathic groove of 14-3-3. This motif is unique among 14-3-3 binding partners, and the precise dynamics of the interaction is not yet fully understood. Here, we investigate this interaction at the molecular level by analyzing the binding of different length p53 CTD peptides to 14-3-3σ using ITC, SPR, NMR, and MD simulations. We observed that the propensity of the p53 peptide to adopt turn-like conformation plays an important role in the binding to the 14-3-3σ protein. Our study contributes to elucidate the molecular mechanism of the 14-3-3-p53 binding and provides useful insight into how conformation properties of a ligand influence protein binding.
Sujet(s)
Protéines 14-3-3/composition chimique , Fragments peptidiques/composition chimique , Protéine p53 suppresseur de tumeur/composition chimique , Séquence d'acides aminés , Sites de fixation , Spectroscopie par résonance magnétique , Simulation de dynamique moléculaire , Liaison aux protéines , Conformation des protéines , Relation structure-activité , Résonance plasmonique de surface , ThermodynamiqueRÉSUMÉ
Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first inâ vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.
Sujet(s)
Antibactériens/pharmacologie , Protéines Escherichia coli/antagonistes et inhibiteurs , Escherichia coli/effets des médicaments et des substances chimiques , Inhibiteurs de la sérine protéinase/pharmacologie , Antibactériens/synthèse chimique , Antibactériens/composition chimique , Relation dose-effet des médicaments , Escherichia coli/métabolisme , Protéines Escherichia coli/métabolisme , Tests de sensibilité microbienne , Simulation de docking moléculaire , Structure moléculaire , Inhibiteurs de la sérine protéinase/synthèse chimique , Inhibiteurs de la sérine protéinase/composition chimique , Relation structure-activitéRÉSUMÉ
RATIONALE: Mass spectrometry (MS) is an invaluable tool for the analysis of proteins. However, the sheer amount of data generated in MS studies demands dedicated data-processing tools that are efficient and require minimal user intervention. METHODS: Utilities for Mass Spectrometry Analysis of Proteins (UMSAP) is a graphical user interface designed for efficient post-processing of MS result files. The software is written in Tcl/Tk and can be used in Windows, OS X or Linux. No third party programs or libraries are required. Currently, UMSAP can process data obtained from proteolytic degradation experiments and generates graphical outputs allowing a straightforward interpretation of statistically relevant results. RESULTS: UMSAP is used here to analyze the proteolytic degradation of glycerophosphoryl diester phosphodiesterase GlpQ by the protein quality control protease DegP. Mass spectrometry was used to monitor proteolysis over time in the absence and presence of a peptidic allosteric activator of DegP. The software's output clearly shows the increased proteolytic activity of DegP in the presence of the activating peptide, identifies statistically significant products of the proteolysis and offers valuable insights into substrate specificity. CONCLUSIONS: Utilities for Mass Spectrometry Analysis of Proteins is an open-source software designed for efficient post-processing of large datasets obtained by MS analyses of proteins. In addition, the modular architecture of the software allows easy incorporation of new modules to analyze various experimental mass spectrometry setups.
Sujet(s)
Spectrométrie de masse/méthodes , Protéines/analyse , Protéomique/méthodes , Logiciel , Bases de données de protéines , Modèles moléculairesRÉSUMÉ
The Aß1-42 dimer is the smallest oligomer of the 42-residue Aß peptide (Aß1-42), which is involved in Alzheimer's disease. The molecular tweezer CLR01 is a synthetic molecule that selectively binds lysine and arginine residues to inhibit toxic aggregation of amyloidogenic peptides. Here, we performed replica exchange molecular dynamics simulations of Aß1-42 in explicit water to study, at the molecular level, the effect of CLR01 binding to the lysine and arginine residues in the dimer. We found that CLR01 molecules encapsulate both lysine residues of each Aß1-42 monomer while only establishing labile interactions with the arginine residues. This encapsulation leads to the noncovalent disruption of inter- and intramolecular interactions involving the monomers. Additionally, the total ß-sheet content in the Aß1-42 dimer decreases because of this binding. With CLR03, a negative control molecule that shares the charged core of CLR01 but does not form inclusion complexes, Aß1-42 dimer formation is observed, similar to the reference system without ligands. Our work allows establishing a molecular mechanism for the modulation of protein-protein interactions in Aß1-42 by CLR01. This mechanism is characterized by an aggregation pathway driven by the encapsulation of lysine residues as well as by the secondary interactions of tweezers with the peptide units and with other CLR01 molecules.
Sujet(s)
Peptides bêta-amyloïdes/antagonistes et inhibiteurs , Peptides bêta-amyloïdes/toxicité , Composés pontés/pharmacologie , Structures macromoléculaires/pharmacologie , Simulation de dynamique moléculaire , Organophosphates/pharmacologie , Fragments peptidiques/antagonistes et inhibiteurs , Fragments peptidiques/toxicité , Peptides bêta-amyloïdes/composition chimique , Composés pontés/composition chimique , Humains , Structures macromoléculaires/composition chimique , Organophosphates/composition chimique , Fragments peptidiques/composition chimique , Relation structure-activitéRÉSUMÉ
Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.
Sujet(s)
Amyloïde/composition chimique , Protéine huntingtine/génétique , Mutation , Peptides/composition chimique , Système acellulaire , Exons , Humains , Protéine huntingtine/composition chimique , Microscopie à force atomique , Microscopie électronique , Modèles moléculaires , Agrégats de protéines , Structure secondaire des protéinesRÉSUMÉ
Protein regions that are involved in protein-protein interactions (PPIs) very often display a high degree of intrinsic disorder, which is reduced during the recognition process. A prime example is binding of the rigid 14-3-3 adapter proteins to their numerous partner proteins, whose recognition motifs undergo an extensive disorder-to-order transition. In this context, it is highly desirable to control this entropy-costly process using tailored stabilizing agents. This study reveals how the molecular tweezer CLR01 tunes the 14-3-3/Cdc25CpS216 protein-protein interaction. Protein crystallography, biophysical affinity determination and biomolecular simulations unanimously deliver a remarkable finding: a supramolecular "Janus" ligand can bind simultaneously to a flexible peptidic PPI recognition motif and to a well-structured adapter protein. This binding fills a gap in the protein-protein interface, "freezes" one of the conformational states of the intrinsically disordered Cdc25C protein partner and enhances the apparent affinity of the interaction. This is the first structural and functional proof of a supramolecular ligand targeting a PPI interface and stabilizing the binding of an intrinsically disordered recognition motif to a rigid partner protein.
Sujet(s)
Protéines 14-3-3/composition chimique , Entropie , Protéines intrinsèquement désordonnées/composition chimique , Ligands , cdc25 Phosphatases/composition chimique , Protéines 14-3-3/métabolisme , Motifs d'acides aminés , Sites de fixation , Protéines intrinsèquement désordonnées/métabolisme , Modèles moléculaires , Peptides/composition chimique , Peptides/métabolisme , Liaison aux protéines , Conformation des protéines , Stabilité protéique , cdc25 Phosphatases/métabolismeRÉSUMÉ
Huntington's disease is a neurodegenerative disorder associated with the expansion of the polyglutamine tract in the exon-1 domain of the huntingtin protein (htte1). Above a threshold of 37 glutamine residues, htte1 starts to aggregate in a nucleation-dependent manner. A 17-residue N-terminal fragment of htte1 (N17) has been suggested to play a crucial role in modulating the aggregation propensity and toxicity of htte1. Here we identify N17 as a potential target for novel therapeutic intervention using the molecular tweezer CLR01. A combination of biochemical experiments and computer simulations shows that binding of CLR01 induces structural rearrangements within the htte1 monomer and inhibits htte1 aggregation, underpinning the key role of N17 in modulating htte1 toxicity.
Sujet(s)
Composés pontés/pharmacologie , Protéine huntingtine/antagonistes et inhibiteurs , Organophosphates/pharmacologie , Composés pontés/composition chimique , Exons , Humains , Interactions hydrophobes et hydrophiles , Simulation de dynamique moléculaire , Structure moléculaire , Organophosphates/composition chimique , Agrégats de protéines/effets des médicaments et des substances chimiquesRÉSUMÉ
The self-organizational properties of DNA have been used to realize synthetic hosts for protein encapsulation. However, current strategies of DNA-protein conjugation still limit true emulation of natural host-guest systems, whose formation relies on non-covalent bonds between geometrically matching interfaces. Here we report one of the largest DNA-protein complexes of semisynthetic origin held in place exclusively by spatially defined supramolecular interactions. Our approach is based on the decoration of the inner surface of a DNA origami hollow structure with multiple ligands converging to their corresponding binding sites on the protein surface with programmable symmetry and range-of-action. Our results demonstrate specific host-guest recognition in a 1:1 stoichiometry and selectivity for the guest whose size guarantees sufficient molecular diffusion preserving short intermolecular distances. DNA nanocontainers can be thus rationally designed to trap single guest molecules in their native form, mimicking natural strategies of molecular recognition and anticipating a new method of protein caging.
Sujet(s)
ADN/composition chimique , Protéines du choc thermique/composition chimique , Ligands , Protéines périplasmiques/composition chimique , Motifs et domaines d'intéraction protéique , Serine endopeptidases/composition chimique , Sites de fixation , Techniques de chimie synthétique , Génie génétique , Protéines du choc thermique/génétique , Modèles moléculaires , Imagerie moléculaire , Sondes moléculaires , Structure moléculaire , Protéines périplasmiques/génétique , Polymères/composition chimique , Serine endopeptidases/génétiqueRÉSUMÉ
Interaction mapping is a powerful strategy to elucidate the biological function of protein assemblies and their regulators. Here, we report the generation of a quantitative interaction network, directly linking 14 human proteins to the AAA+ ATPase p97, an essential hexameric protein with multiple cellular functions. We show that the high-affinity interacting protein ASPL efficiently promotes p97 hexamer disassembly, resulting in the formation of stable p97:ASPL heterotetramers. High-resolution structural and biochemical studies indicate that an extended UBX domain (eUBX) in ASPL is critical for p97 hexamer disassembly and facilitates the assembly of p97:ASPL heterotetramers. This spontaneous process is accompanied by a reorientation of the D2 ATPase domain in p97 and a loss of its activity. Finally, we demonstrate that overproduction of ASPL disrupts p97 hexamer function in ERAD and that engineered eUBX polypeptides can induce cell death, providing a rationale for developing anti-cancer polypeptide inhibitors that may target p97 activity.
Sujet(s)
Dégradation associée au réticulum endoplasmique/physiologie , Protéines de fusion oncogènes/métabolisme , Domaines protéiques/physiologie , Protéine contenant la valosine/métabolisme , Encéphale/anatomopathologie , Prolifération cellulaire , Cristallographie aux rayons X , Cellules HEK293 , Humains , Protéines et peptides de signalisation intracellulaire , Mutation , Protéines de fusion oncogènes/composition chimique , Protéines de fusion oncogènes/isolement et purification , Peptides/génétique , Peptides/métabolisme , Liaison aux protéines , Ingénierie des protéines , Cartes d'interactions protéiques , Multimérisation de protéines , Structure quaternaire des protéines , Protéines recombinantes/composition chimique , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Protéine contenant la valosine/composition chimique , Protéine contenant la valosine/isolement et purificationRÉSUMÉ
Enzyme-directed mutasynthesis is an emerging strategy for the targeted derivatization of natural products. Here, data on the synthesis of malonic acid derivatives for feeding studies in Saccharopolyspora erythraea , the mutagenesis of DEBS and bioanalytical data on the experimental investigation of studies on the biosynthetic pathway towards erythromycin are presented.
RÉSUMÉ
Polyketides are natural products frequently used for the treatment of various diseases, but their structural complexity hinders efficient derivatization. In this context, we recently introduced enzyme-directed mutasynthesis to incorporate non-native extender units into the biosynthesis of erythromycin. Modeling and mutagenesis studies led to the discovery of a variant of an acyltransferase domain in the erythromycin polyketide synthase capable of accepting a propargylated substrate. Here, we extend molecular rationalization of enzyme-substrate interactions through modeling, to investigate the incorporation of substrates with different degrees of saturation of the malonic acid side chain. This allowed the engineered biosynthesis of new erythromycin derivatives and the introduction of additional mutations into the AT domain for a further shift of the enzyme's substrate scope. Our approach yields non-native polyketide structures with functional groups that will simplify future derivatization approaches, and provides a blueprint for the engineering of AT domains to achieve efficient polyketide synthase diversification.
Sujet(s)
Acyltransferases/métabolisme , Polycétides/métabolisme , Acyltransferases/génétique , Antibactériens/composition chimique , Antibactériens/métabolisme , Érythromycine/analogues et dérivés , Érythromycine/biosynthèse , Simulation de dynamique moléculaire , Mutagenèse dirigée , Polyketide synthases/composition chimique , Structure tertiaire des protéines , Spécificité du substratRÉSUMÉ
Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.
Sujet(s)
Amyloïde/antagonistes et inhibiteurs , Agents antiVIH/pharmacologie , Antimétabolites/pharmacologie , Composés pontés/pharmacologie , Organophosphates/pharmacologie , Sperme/effets des médicaments et des substances chimiques , Transmission de maladie infectieuse/prévention et contrôle , Infections à VIH/prévention et contrôle , Infections à VIH/transmission , Humains , Mâle , Sperme/composition chimique , Sperme/virologieRÉSUMÉ
In type-2 diabetes (T2D), islet amyloid polypeptide (IAPP) self-associates into toxic assemblies causing islet ß-cell death. Therefore, preventing IAPP toxicity is a promising therapeutic strategy for T2D. The molecular tweezer CLR01 is a supramolecular tool for selective complexation of K residues in (poly)peptides. Surprisingly, it inhibits IAPP aggregation at substoichiometric concentrations even though IAPP has only one K residue at position 1, whereas efficient inhibition of IAPP toxicity requires excess CLR01. The basis for this peculiar behavior is not clear. Here, a combination of biochemical, biophysical, spectroscopic, and computational methods reveals a detailed mechanistic picture of the unique dual inhibition mechanism for CLR01. At low concentrations, CLR01 binds to K1, presumably nucleating nonamyloidogenic, yet toxic, structures, whereas excess CLR01 binds also to R11, leading to nontoxic structures. Encouragingly, the CLR01 concentrations needed for inhibition of IAPP toxicity are safe in vivo, supporting its development toward disease-modifying therapy for T2D.
Sujet(s)
Composés pontés/composition chimique , Hypoglycémiants/composition chimique , Cellules à insuline/effets des médicaments et des substances chimiques , Polypeptide amyloïde des ilots/antagonistes et inhibiteurs , Organophosphates/composition chimique , Animaux , Composés pontés/pharmacologie , Lignée cellulaire , Survie cellulaire/effets des médicaments et des substances chimiques , Humains , Hypoglycémiants/pharmacologie , Cellules à insuline/cytologie , Cellules à insuline/métabolisme , Polypeptide amyloïde des ilots/composition chimique , Polypeptide amyloïde des ilots/toxicité , Modèles moléculaires , Organophosphates/pharmacologie , Agrégats de protéines/effets des médicaments et des substances chimiques , Liaison aux protéines , Structure secondaire des protéines , Structure tertiaire des protéines , RatsRÉSUMÉ
The benzyl radical (1) is a key intermediate in the combustion and tropospheric oxidation of toluene. Because of its relevance, the reaction of 1 with molecular oxygen was investigated by matrix-isolation IR and EPR spectroscopy as well as computational methods. The primary reaction product of 1 and O2 is the benzylperoxyl radical (2), which exists in several conformers that can easily interconvert even at cryogenic temperatures. Photolysis of radical 2 at 365â nm results in a formal [1,3]-H migration and subsequent cleavage of the O-O bond to produce a hydrogen-bonded complex between the hydroxyl radical and benzaldehyde (4). Prolonged photolysis produces the benzoyl radical (5) and water, which finally yield the phenyl radical (7), CO, and H2O. Thus, via a sequence of exothermic reactions 1 is transformed into radicals of even higher reactivity, such as OH and 7. Our results have implications for the development of models for the highly complicated process of combustion of aromatic compounds.
RÉSUMÉ
The polyether ionophore monensin is biosynthesized by a polyketide synthase that delivers a mixture of monensins A and B by the incorporation of ethyl- or methyl-malonyl-CoA at its fifth module. Here we present the first computational model of the fifth acyltransferase domain (AT5mon ) of this polyketide synthase, thus affording an investigation of the basis of the relaxed specificity in AT5mon , insights into the activation for the nucleophilic attack on the substrate, and prediction of the incorporation of synthetic malonic acid building blocks by this enzyme. Our predictions are supported by experimental studies, including the isolation of a predicted derivative of the monensin precursor premonensin. The incorporation of non-native building blocks was found to alter the ratio of premonensins A and B. The bioactivity of the natural product derivatives was investigated and revealed binding to prenyl-binding protein. We thus show the potential of engineered biosynthetic polyketides as a source of ligands for biological macromolecules.
Sujet(s)
Produits biologiques/synthèse chimique , Monensin/analogues et dérivés , Monensin/synthèse chimique , Polyketide synthases/composition chimique , Acyltransferases/composition chimique , Biologie informatique , Escherichia coli/métabolisme , Fermentation , Malonates/composition chimique , Modèles moléculaires , Monensin/pharmacologie , Conformation des protéines , Streptomyces/enzymologie , Spécificité du substratRÉSUMÉ
The three-dimensional structure of a peptide is strongly influenced by its solvent environment. In the present study, we study three cyclic tetrapeptides which serve as model peptides for ß-turns. They are of the general structure cyclo(Boc-Cys-Pro-X-Cys-OMe) with the amino acid X being either glycine (1), or L- or D-leucine (L- or D-2). Using vibrational circular dichroism (VCD) spectroscopy, we confirm previous NMR results which showed that D-2 adopts predominantly a ßII turn structure in apolar and polar solvents. Our results for L-2 indicate a preference for a ßI structure over ßII. With increasing solvent polarity, the preference for 1 is shifted from ßII towards ßI. This conformational change goes along with the breaking of an intramolecular hydrogen bond which stabilizes the ßII conformation. Instead, a hydrogen bond with a solvent molecule can stabilize the ßI turn conformation.