RESUMEN
Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.
RESUMEN
The COVID-19 pandemic represents a global challenge that has impacted and is expected to continue to impact the lives and health of people across the world for the foreseeable future. The rollout of vaccines has provided highly anticipated relief, but effective therapeutics are required to further reduce the risk and severity of infections. Monoclonal antibodies have been shown to be effective as therapeutics for SARS-CoV-2, but as new variants of concern (VoC) continue to emerge, their utility and use have waned due to limited or no efficacy against these variants. Furthermore, cumbersome systemic administration limits easy and broad access to such drugs. As well, concentrations of systemically administered antibodies in the mucosal epithelium, a primary site of initial infection, are dependent on neonatal Fc receptor mediated transport and require high drug concentrations. To reduce the viral load more effectively in the lung, we developed an inhalable formulation of a SARS-CoV-2 neutralizing antibody binding to a conserved epitope on the Spike protein, ensuring pan-neutralizing properties. Administration of this antibody via a vibrating mesh nebulization device retained antibody integrity and resulted in effective distribution of the antibody in the upper and lower respiratory tract of non-human primates (NHP). In comparison with intravenous administration, significantly higher antibody concentrations can be obtained in the lung, resulting in highly effective reduction in viral load post SARS-CoV-2 challenge. This approach may reduce the barriers of access and uptake of antibody therapeutics in real-world clinical settings and provide a more effective blueprint for targeting existing and potentially emerging respiratory tract viruses.
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Antivirales , COVID-19 , Animales , Humanos , SARS-CoV-2 , Pandemias , Anticuerpos Antivirales , Anticuerpos Neutralizantes , Epítopos , Glicoproteína de la Espiga del CoronavirusRESUMEN
Hydrogen-deuterium eXchange mass spectrometry (HDX-MS) is increasingly used in drug development to locate binding sites and to identify allosteric effects in drug/target interactions. However, the potential of this technique to quantitatively analyze drug candidate libraries remains largely unexplored. Here, a collection of 13 WDR5-targeting small molecules with surface plasmon resonance (SPR) dissociation coefficients (KD) ranging from 20 nM to â¼116 µM were characterized using differential HDX-MS (ΔHDX-MS). Conventional qualitative analysis of the ΔHDX-MS data set revealed the binding interfaces for all compounds and allosteric effects where present. We then demonstrated that ΔHDX-MS signal-to-noise (S/N) not only can rank library-relative affinity but also can accurately predict KD from a calibration curve constructed from high-quality SPR data. Three methods for S/N calculation are explored, each suitable for libraries with different characteristics. Our results demonstrate the potential for ΔHDX-MS use in drug candidate library affinity validation and/or determination while simultaneously characterizing structure.
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Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Bibliotecas de Moléculas Pequeñas , Resonancia por Plasmón de Superficie , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio/métodos , Resonancia por Plasmón de Superficie/métodos , HumanosRESUMEN
Life at the molecular level is a dynamic world, where the key playersâproteins, oligonucleotides, lipids, and carbohydratesâare in a perpetual state of structural flux, shifting rapidly between local minima on their conformational free energy landscapes. The techniques of classical structural biology, X-ray crystallography, structural NMR, and cryo-electron microscopy (cryo-EM), while capable of extraordinary structural resolution, are innately ill-suited to characterize biomolecules in their dynamically active states. Subsecond time-resolved mass spectrometry (MS) provides a unique window into the dynamic world of biological macromolecules, offering the capacity to directly monitor biochemical processes and conformational shifts with a structural dimension provided by the electrospray charge-state distribution, ion mobility, covalent labeling, or hydrogen-deuterium exchange. Over the past two decades, this suite of techniques has provided important insights into the inherently dynamic processes that drive function and pathogenesis in biological macromolecules, including (mis)folding, complexation, aggregation, ligand binding, and enzyme catalysis, among others. This Review provides a comprehensive account of subsecond time-resolved MS and the advances it has enabled in dynamic structural biology, with an emphasis on insights into the dynamic drivers of protein function.
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Biología , Proteínas , Microscopía por Crioelectrón/métodos , Espectrometría de Masas/métodos , Conformación Proteica , Proteínas/químicaRESUMEN
Based on the mimicry of microbial metabolites, functionalized indoles were demonstrated as the ligands and agonists of the pregnane X receptor (PXR). The lead indole, FKK6, displayed PXR-dependent protective effects in DSS-induced colitis in mice and in vitro cytokine-treated intestinal organoid cultures. Here, we report on the initial in vitro pharmacological profiling of FKK6. FKK6-PXR interactions were characterized by hydrogen-deuterium exchange mass spectrometry. Screening FKK6 against potential cellular off-targets (G protein-coupled receptors, steroid and nuclear receptors, ion channels, and xenobiotic membrane transporters) revealed high PXR selectivity. FKK6 has poor aqueous solubility but was highly soluble in simulated gastric and intestinal fluids. A large fraction of FKK6 was bound to plasma proteins and chemically stable in plasma. The partition coefficient of FKK6 was 2.70, and FKK6 moderately partitioned into red blood cells. In Caco2 cells, FKK6 displayed high permeability (A-B: 22.8 × 10-6 cm.s-1) and no active efflux. These data are indicative of essentially complete in vivo absorption of FKK6. The data from human liver microsomes indicated that FKK6 is rapidly metabolized by cytochromes P450 (t1/2 5 min), notably by CYP3A4. Two oxidized FKK6 derivatives, including DC73 (N6-oxide) and DC97 (C19-phenol), were detected, and these metabolites had 5-7 × lower potency as PXR agonists than FKK6. This implies that despite high intestinal absorption, FKK6 is rapidly eliminated by the liver, and its PXR effects are predicted to be predominantly in the intestines. In conclusion, the PXR ligand and agonist FKK6 has a suitable pharmacological profile supporting its potential preclinical development.
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Colitis , Humanos , Animales , Ratones , Receptor X de Pregnano/agonistas , Células CACO-2 , Colitis/inducido químicamente , Receptores Citoplasmáticos y Nucleares , Antiinflamatorios/uso terapéuticoRESUMEN
The structurally conserved B-cell lymphoma 2 (Bcl-2) family of protein function to promote or inhibit apoptosis through an exceedingly complex web of specific, intrafamilial protein-protein interactions. The critical role of these proteins in lymphomas and other cancers has motivated a widespread interest in understanding the molecular mechanisms that drive specificity in Bcl-2 family interactions. However, the high degree of structural similarity among Bcl-2 homologues has made it difficult to rationalize the highly specific (and often divergent) binding behavior exhibited by these proteins using conventional structural arguments. In this work, we use time-resolved hydrogen deuterium exchange mass spectrometry to explore shifts in conformational dynamics associated with binding partner engagement in the Bcl-2 family proteins Bcl-2 and Mcl-1. Using this approach combined with homology modeling, we reveal that Mcl-1 binding is driven by a large-scale shift in conformational dynamics, while Bcl-2 complexation occurs primarily through a classical charge compensation mechanism. This work has implications for understanding the evolution of internally regulated biological systems composed of structurally similar proteins and for the development of drugs targeting Bcl-2 family proteins for promotion of apoptosis in cancer.
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Proteínas Reguladoras de la Apoptosis , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Unión Proteica , ApoptosisRESUMEN
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a rapidly growing technique for protein characterization in industry and academia, complementing the "static" picture provided by classical structural biology with information about the dynamic structural changes that accompany biological function. Conventional hydrogen deuterium exchange experiments, carried out on commercially available systems, typically collect 4-5 exchange timepoints on a timescale ranging from tens of seconds to hours using a workflow that can require 24 h or more of continuous data collection for triplicate measurements. A small number of groups have developed setups for millisecond timescale HDX, allowing for the characterization of dynamic shifts in weakly structured or disordered regions of proteins. This capability is particularly important given the central role that weakly ordered protein regions often play in protein function and pathogenesis. In this work, we introduce a new continuous flow injection setup for time-resolved HDX-MS (CFI-TRESI-HDX) that allows automated, continuous or discrete labeling time measurements from milliseconds to hours. The device is composed almost entirely of "off-the-shelf" LC components and can acquire an essentially unlimited number of timepoints with substantially reduced runtimes compared to conventional systems.
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Medición de Intercambio de Deuterio , Tetranitrato de Pentaeritritol , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Recolección de Datos , HidrógenoRESUMEN
α-Carboxyketose synthases, including 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS), are long-standing targets for inhibition. They are challenging targets to create tight-binding inhibitors against, and inhibitors often display half-of-sites binding and partial inhibition. Half-of-sites inhibition demonstrates the existence of inter-subunit communication in DAHPS. We used X-ray crystallography and spatially resolved hydrogen-deuterium exchange (HDX) to reveal the structural and dynamic bases for inter-subunit communication in Escherichia coli DAHPS(Phe), the isozyme that is feedback-inhibited by phenylalanine. Crystal structures of this homotetrameric (dimer-of-dimers) enzyme are invariant over 91% of its sequence. Three variable loops make up 8% of the sequence and are all involved in inter-subunit contacts across the tight-dimer interface. The structures have pseudo-twofold symmetry indicative of inter-subunit communication across the loose-dimer interface, with the diagonal subunits B and C always having the same conformation as each other, while subunits A and D are variable. Spatially resolved HDX reveals contrasting responses to ligand binding, which, in turn, affect binding of the second substrate, erythrose-4-phosphate (E4P). The N-terminal peptide, M1-E12, and the active site loop that binds E4P, F95-K105, are key parts of the communication network. Inter-subunit communication appears to have a catalytic role in all α-carboxyketose synthase families and a regulatory role in some members.
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3-Desoxi-7-Fosfoheptulonato Sintasa , Isoenzimas , 3-Desoxi-7-Fosfoheptulonato Sintasa/química , Sitios de Unión , Catálisis , Comunicación , Cristalografía por Rayos X , Deuterio , Escherichia coli , Humanos , Isoenzimas/metabolismo , Ligandos , Fenilalanina/metabolismo , FosfatosRESUMEN
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.
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Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Análisis de Datos , Concentración de Iones de HidrógenoRESUMEN
Consuming sugar-sweetened beverages is one of the main concerns in addressing obesity in North Carolina. Many interventions have been developed to limit their consumptionwhile promoting drinking more water. Major sugarsweetened beverage producers take no accountability for the harm they cause to communities.
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Bebidas Azucaradas , Bebidas/efectos adversos , Humanos , North Carolina , Obesidad/etiología , Obesidad/prevención & control , Bebidas Azucaradas/efectos adversosRESUMEN
Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.
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Lipocalina 2/fisiología , Mitocondrias/patología , Miocitos Cardíacos/patología , Especies Reactivas de Oxígeno/metabolismo , Daño por Reperfusión/patología , Sideróforos/metabolismo , Animales , Gentisatos/farmacología , Hidroxibenzoatos/farmacología , Hierro/metabolismo , Masculino , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fosforilación Oxidativa , Ratas , Ratas Wistar , Daño por Reperfusión/tratamiento farmacológico , Daño por Reperfusión/metabolismoRESUMEN
INTRODUCTION: Distal radius fractures are extremely common injuries affecting a wide range of patient demographics. The purpose of this study was to evaluate the outcomes of distal radius fractures managed initially with closed reduction and immobilization in either a below elbow volar-dorsal splint versus sugar-tong splint prior to conversion into a short arm cast. METHODS: We performed a retrospective study of patients with distal radius fractures placed in a sugar-tong (n = 45) and volar-dorsal splint (n = 36). Anteroposterior and lateral radiographs were evaluated immediately after closed reduction and placement into either a sugar-tong or volar-dorsal splint. The radial inclination, radial length, volar tilt, and intra-articular displacement were measured. RESULTS: The average age was not significantly different between groups (Diff: 1.1 years, P = 0.8766). Initial clinic follow-up radiographs illustrated significantly lower radial inclination in the sugar-tong group than volar-dorsal group (17.1 vs. 19, P = 0.0443). Follow-up mean radial length was not significantly lower in the sugar-tong than volar-dorsal group (8.4 vs. 9.2, P = 0.0858). Palmar tilt and articular step-off was not significantly different between splint types. The loss of reduction was 28.8% for the sugar-tong and 25.0% for the volar-dorsal group (P = 0.696). CONCLUSION: Our results did not demonstrate a significant difference in loss of reduction rates between the two splint groups. There was no significant difference between the sugar-tong and volar-dorsal groups in terms of loss of radial length and volar tilt. Loss of reduction was similar between groups suggesting no advantage of a volar-dorsal splint compared to a sugar-tong splint. LEVEL OF EVIDENCE: Therapeutic level III.
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Fracturas del Radio , Férulas (Fijadores) , Adulto , Placas Óseas , Fijación Interna de Fracturas , Humanos , Lactante , Radiografía , Fracturas del Radio/diagnóstico por imagen , Fracturas del Radio/terapia , Rango del Movimiento Articular , Estudios Retrospectivos , Azúcares , Resultado del TratamientoRESUMEN
The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen-deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.
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Bevacizumab/inmunología , Biosimilares Farmacéuticos/farmacología , Mapeo Epitopo , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Factor A de Crecimiento Endotelial Vascular/química , Humanos , Cinética , Microfluídica , Modelos MolecularesRESUMEN
La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3'OH-tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3'OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen-deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein-protein interdomain interaction in La proteins.
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Núcleo Celular/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Motivo de Reconocimiento de ARN , ARN/metabolismo , Sitios de Unión , Núcleo Celular/genética , Humanos , Modelos Moleculares , Mutación , Fosfoproteínas/genética , Unión Proteica , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , ARN/químicaRESUMEN
Primarily known for its function in the electron transport chain, cytochrome c (Cyt c) also plays a critical role in the initiation of mitochondrially induced apoptosis through specific interactions with cardiolipin (CL), a negatively charged phospholipid found in the inner mitochondrial membrane. In this work, we study the conformational dynamics of Cyt c in the presence of CL and phosphatidylcholine (PC) phospholipids also present in the mitochondrial membrane to better understand how these interactions might drive transformation to the peroxidase-active protein. Using ion mobility mass spectrometry and millisecond hydrogen-deuterium exchange mass spectrometry, we demonstrate heterogeneity in the lipid-bound ensemble, with zwitterionic (PC) phospholipids inducing destabilization of residues necessary for peroxidase coordination, and increased dynamics on the proximal face of the heme binding pocket. In contrast to what might be expected from classical models for CL-driven Cyt c peroxidase activation, interactions with CL are shown to rigidify heme coordination. To reconcile this observation with the well-supported view that CL is linked to peroxidase activation, we propose a mechanism in which CL stabilizes the conformational transition between the peroxidase-active and inactive forms.
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Cardiolipinas/metabolismo , Citocromos c/metabolismo , Fosfatidilcolinas/metabolismo , Animales , Caballos/metabolismo , Conformación ProteicaRESUMEN
The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Toxina Diftérica/inmunología , Epítopos/inmunología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Neutralizantes/metabolismo , Corynebacterium diphtheriae/química , Deuterio/química , Medición de Intercambio de Deuterio , Toxina Diftérica/química , Toxina Diftérica/metabolismo , Mapeo Epitopo , Epítopos/metabolismo , Cinética , NAD/metabolismo , Unión Proteica/inmunología , Conformación Proteica , Dominios Proteicos/inmunologíaRESUMEN
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25â¯years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2O) for a variable 'labeling time', resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired. When coupled with MS, HDX is suitable for probing allosteric effects in catalysis and ligand binding, epitope mapping, validation of biosimilars, drug candidate screening and mapping membrane-protein interactions among many other bioanalytical applications. This review introduces HDX-MS via a brief description of HDX-MS development, followed by an overview of HDX theory and ultimately an outline of methods and procedures involved in performing HDX-MS experiments.
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Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Mapeo Epitopo , Humanos , Ligandos , Conformación Proteica , Mapeo de Interacción de Proteínas , Proteínas/química , Proteínas/metabolismoRESUMEN
The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g., by abrogating or permitting new charge-charge interactions, the basis for enhancement of the hydrophobically driven interactions that are typical of disordered protein-target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal transactivation domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using time-resolved electrospray ionization with hydrogen-deuterium exchange, we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to the PH domain but instead increases the degree of conformational disorder in the unbound state. We propose that this increase in the degree of disorder creates a wider free energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53-target complexes have similar free energies.
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Proteína p53 Supresora de Tumor/química , Medición de Intercambio de Deuterio , Humanos , Dominios Homólogos a Pleckstrina , Unión Proteica , Estabilidad Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the "domain rotation model" suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the "rigid body reorientation model" proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the ß-strands within the (ß/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.
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Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Modelos Moleculares , Piruvato Quinasa/química , Regulación Alostérica/fisiología , Medición de Intercambio de Deuterio , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Piruvato Quinasa/genéticaRESUMEN
Many important chemical and biochemical phenomena proceed on sub-second time scales before entering equilibrium. In this mini-review, we explore the history and recent advancements of time-resolved mass spectrometry (TRMS) for the characterization of millisecond time-scale chemical reactions and biochemical processes. TRMS allows for the simultaneous tracking of multiple reactants, intermediates and products with no chromophoric species required, high sensitivity and temporal resolution. The method has most recently been used for the characterization of several short-lived reaction intermediates in rapid chemical reactions. Most of the reactions that occur in living organisms are accelerated by enzymes, with pre-steady state kinetics only attainable using time-resolved methods. TRMS has been increasingly used to monitor the conversion of substrates to products and the resulting changes to the enzyme during catalytic turnover. Early events in protein folding systems have also been elucidated, along with the characterization of dynamics and transient secondary structures in intrinsically disordered proteins. In this review, we will highlight representative examples where TRMS has been applied to study these phenomena.