ABSTRACT
The collaborative total synthesis of darobactin A, a recently isolated antibiotic that selectively targets Gram-negative bacteria, has been accomplished in a convergent fashion with a longest linear sequence of 16 steps from d-Garner's aldehyde and l-serine. Scalable routes toward three non-canonical amino acids were developed to enable the synthesis. The closure of the bismacrocycle was realized through sequential, halogen-selective Larock indole syntheses, where the proper order of cyclizations proved crucial for the formation of the desired atropisomer of the natural product.
Subject(s)
Aldehydes , Amino Acids , Aldehydes/chemistry , Amino Acids/chemistry , Cyclization , Phenylpropionates , StereoisomerismABSTRACT
Recent advances in the field of cancer biology have accelerated the discovery and development of novel biopharmaceuticals. At the forefront of these drug development efforts are high-throughput screening, compressed timelines, and limited sample quantities, all characteristic of the discovery space. To meet program targets, large numbers of protein variants must be produced, screened, and characterized, presenting a daunting analytical challenge. Additionally, the higher-order structure is paramount for protein function and must be monitored as a critical quality attribute. Matrix-assisted laser desorption/ionization mass spectrometry has been utilized as an ultra-fast, automatable, sample-sparing analytical tool for biomolecules. Our group has published applications integrating hydrogen-deuterium exchange mass spectrometry with matrix-assisted laser desorption/ionization mass spectrometry for the rapid conformational characterization of small proteins, the current work expands this application to monoclonal and bi-specific antibodies. This study demonstrates the ability of the methodology, matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry, to detect conformational differences between bi-specific antibodies from different expression hosts. These conformational differences were validated by orthogonal techniques including circular dichroism, nuclear magnetic resonance, and size-exclusion chromatography hydrogen-deuterium exchange mass spectrometry. This work demonstrates the utility of applying the developed methodology as a rapid conformational screening tool to triage samples for further analytical characterization.
Subject(s)
Deuterium Exchange Measurement , Hydrogen , Deuterium/chemistry , Deuterium/metabolism , Deuterium Exchange Measurement/methods , Hydrogen/chemistry , Lasers , Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Tightly regulated activity of the transcription factor MYC is essential for orderly cell proliferation. Upon deregulation, MYC elicits and promotes cancer progression. Proteasomal degradation is an essential element of MYC regulation, initiated by phosphorylation at Serine62 (Ser62) of the MB1 region. Here, we found that Ser62 phosphorylation peaks in mitosis, but that a fraction of nonphosphorylated MYC binds to the microtubules of the mitotic spindle. Consequently, the microtubule-destabilizing drug vincristine decreases wild-type MYC stability, whereas phosphorylation-deficient MYC is more stable, contributing to vincristine resistance and induction of polyploidy. PI3K inhibition attenuates postmitotic MYC formation and augments the cytotoxic effect of vincristine. IMPLICATIONS: The spindle's function as a docking site for MYC during mitosis may constitute a window of specific vulnerability to be exploited for cancer treatment.
Subject(s)
Biomarkers, Tumor/metabolism , Gene Expression Regulation, Neoplastic , Microtubules/metabolism , Mitosis , Neoplasms/pathology , Proto-Oncogene Proteins c-myc/metabolism , Vincristine/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis , Biomarkers, Tumor/genetics , Cell Cycle , Cell Proliferation , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-myc/genetics , Tumor Cells, CulturedABSTRACT
Förster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with B 0 2 -dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.1 GHz. These structural data provide the unprecedented opportunity to calculate FRET from the x-ray structure and to compare it to experimental data in solution. We find that interdomain dynamics limits the FRET effect and show that a rigidification of the sensor further enhances FRET.
Subject(s)
Biosensing Techniques/methods , Green Fluorescent Proteins/chemistry , Fluorescence Resonance Energy Transfer , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Scattering, Small Angle , X-Ray DiffractionABSTRACT
Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.
Subject(s)
Guanylate Kinases/metabolism , Allosteric Regulation , Animals , Cell Line, Tumor , Crystallography, X-Ray , Guanylate Kinases/chemistry , Guanylate Kinases/genetics , Humans , Kinetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purificationABSTRACT
Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain 1H, 13C, and 15N nuclei as a first step towards the enzyme's structural and mechanistic analysis with atomic resolution.
Subject(s)
Guanylate Kinases/chemistry , Nuclear Magnetic Resonance, Biomolecular , HumansABSTRACT
Understanding the dissociation of molecules is the basis to modulate interactions of biomedical interest. Optimizing drugs for dissociation rates is found to be important for their efficacy, selectivity, and safety. Here, we show an application of the high-power relaxation dispersion (RD) method to the determination of the dissociation rates of weak binding ligands from receptors. The experiment probes proton RD on the ligand and, therefore, avoids the need for any isotopic labeling. The large ligand excess eases the detection significantly. Importantly, the use of large spin-lock fields allows the detection of faster dissociation rates than other relaxation approaches. Moreover, this experimental approach allows to access directly the off-rate of the binding process without the need for analyzing a series of samples with increasing ligand saturation. The validity of the method is shown with small molecule interactions using two macromolecules, bovine serum albumin and tubulin heterodimers.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Kinetics , Protein BindingABSTRACT
The UVA (350 nm) irradiation of an α-pyran in the presence of oxygen led to the unexpected formation of a tetraoxygenated compound whose structure could not be unambiguously determined on the basis of conventional (1)H-(13)C correlated experiments. 1,1-ADEQUATE (adequate double quantum transfer experiment) and 1,n-ADEQUATE combined with computer-assisted structure elucidation software led to two structural possibilities involving the formation of either an epoxide or an oxetane. Residual dipolar couplings allowed not only the identification of the compound as a spiroepoxide but also the determination of its relative configuration. To account for its formation, we propose a bisepoxide intermediate that, as opposed to most α,ß-epoxyketones under irradiation, undergoes O-Cß cleavage probably due to the presence of an extra oxygen substituent in the ß position. 1,2-Acyl migration would then proceed stereoselectively to the final product obtained as a single diastereomer.
ABSTRACT
Lectins from different sources have been shown to interfere with HIV infection by binding to the sugars of viral-envelope glycoproteins. Three-dimensional atomic structures of a number of HIV-inactivating lectins have been determined, both as free proteins and in glycan-bound forms. However, details on the mechanism of recognition and binding to sugars are elusive. Herein we focus on the anti-HIV lectin OAA from Oscillatoria agardhii: We show that in the absence of sugars in solution, both the sugar-free and sugar-bound protein conformations that were observed in the X-ray crystal structures exist as conformational substates. Our results suggest that glycan recognition occurs by conformational selection within the ground state; this model differs from the popular "excited-state" model. Our findings provide further insight into molecular recognition of the major receptor on the HIV virus by OAA. These details can potentially be used for the optimization and/or development of preventive anti-HIV therapeutics.
Subject(s)
Anti-HIV Agents/chemistry , Bacterial Proteins/chemistry , Carbohydrates/chemistry , Lectins/chemistry , Oscillatoria/metabolism , Polysaccharides/chemistry , Anti-HIV Agents/metabolism , Bacterial Proteins/metabolism , Binding Sites , Crystallography, X-Ray , HIV/metabolism , Lectins/metabolism , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Structure, Tertiary , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolismABSTRACT
The conformational state of 8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (lorcaserin) in water has been determined on the basis of one-bond and long-range C-H residual dipolar coupling (RDC) data along with DFT computations and (3)J(HH) coupling-constant analysis. According to this analysis, lorcaserin exists as a conformational equilibrium of two crown-chair forms, of which the preferred conformation has the methyl group in an equatorial orientation.
Subject(s)
Anti-Obesity Agents/chemistry , Benzazepines/chemistry , Water/chemistry , Anti-Obesity Agents/pharmacology , Hydrogen Bonding , Molecular Conformation , Nuclear Magnetic Resonance, BiomolecularABSTRACT
Deuterium NMR imaging was used to evaluate the spatial distribution of the degree of alignment in different types of alignment media by monitoring the deuterium quadrupolar splitting using spatially resolved NMR techniques in conventional liquid state NMR instruments. These images allow the unambiguous distinction of magnetic field and alignment inhomogeneities present in partially aligned samples, revealing the underlying reasons for linebroadening within an alignment medium that cannot be explained by the sole analysis of 1D (2)H NMR spectra. For example, alignment inhomogeneities due to broken gels or the presence of concentration gradients in liquid crystalline solutions are clearly detected by the imaging methods proposed in this work.
Subject(s)
Deuterium/chemistry , Magnetic Resonance Spectroscopy , Gels/chemistry , Liquid Crystals/chemistry , Magnetic Resonance ImagingABSTRACT
19-OH-(-)-eburnamonine 1 is a new indole alkaloid isolated from Bonafusia macrocalyx. A natural derivative from (-)-eburnamonine for which absolute configuration and conformation has been determined by making use of residual dipolar couplings enhanced NMR, circular dichroism spectra and high-level computations.
Subject(s)
Nuclear Magnetic Resonance, Biomolecular/methods , Vinca Alkaloids/chemistry , Models, Molecular , Molecular ConformationABSTRACT
The dependence of molecular alignment with solvent nature and salt concentration has been investigated for mechanically stretched polyacrylamide copolymer gels. Residual dipolar couplings (RDCs) were recorded for D(2)O, DMSO-d(6), and DMSO-d(6)/D(2)O solutions containing different proportions of the solvents and different sodium chloride concentrations. Alignment tensors were determined by fitting the experimental RDCs to the DFT-computed structure of N-methylcodeinium ion. Analysis of the tensors shows that the degree of alignment decreases with the proportion of DMSO-d(6) as well as with the concentration of sodium chloride, most likely due to enhanced ion-pair aggregation. Furthermore, rotation of the alignment tensor is observed when increasing the salt concentration.
Subject(s)
Acrylamide/chemistry , Algorithms , Nuclear Magnetic Resonance, Biomolecular , Polymers/chemistry , Sodium Chloride/chemistry , Deuterium Oxide/chemistry , Dimethyl Sulfoxide/chemistry , Gels/chemistry , Quantum Theory , Solvents/chemistryABSTRACT
The use of the cetylpyridinium chloride (CPCL)/NaCl/hexanol liquid crystal allowed the measurement of (1)D(CH) residual dipolar couplings of the isoquinoline alkaloid salsolidine in its protonated state. Populations of its two half-chair forms were determined by using the single alignment tensor approximation combined with global superposition of conformers. These populations were in good agreement with the DFT-computed energies for both conformers.