RESUMEN
Beta-arrestins (ßarrs) are key regulators and transducers of G-protein coupled receptor signaling; however, little is known of how ßarrs communicate with their downstream effectors. Here, we use cryo-electron microscopy to elucidate how ßarr1 recruits and activates non-receptor tyrosine kinase Src. ßarr1 binds Src SH3 domain via two distinct sites: a polyproline site in the N-domain and a non-proline site in the central crest region. At both sites ßarr1 interacts with the aromatic surface of SH3 which is critical for Src autoinhibition, suggesting that ßarr1 activates Src by SH3 domain displacement. Binding of SH3 to the central crest region induces structural rearrangements in the ß-strand V, finger, and middle loops of ßarr1 and interferes with ßarr1 coupling to the receptor core potentially impacting receptor desensitization and downstream signaling.
RESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site versus allosteric small-molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active-site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX-MS for elucidating aspects of protein conformational dynamics and allosteric effects of small-molecule ligands and highlights the potential of integrating HDX-MS alongside other complementary methods, such as room-temperature X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics.
Asunto(s)
Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Regulación Alostérica , Humanos , Simulación de Dinámica Molecular , Conformación Proteica , Modelos Moleculares , Dominio CatalíticoRESUMEN
Glia derived secretory factors play diverse roles in supporting the development, physiology, and stress responses of the central nervous system (CNS). Through transcriptomics and imaging analyses, we have identified Obp44a as one of the most abundantly produced secretory proteins from Drosophila CNS glia. Protein structure homology modeling and Nuclear Magnetic Resonance (NMR) experiments reveal Obp44a as a fatty acid binding protein (FABP) with a high affinity towards long-chain fatty acids in both native and oxidized forms. Further analyses demonstrate that Obp44a effectively infiltrates the neuropil, traffics between neuron and glia, and is secreted into hemolymph, acting as a lipid chaperone and scavenger to regulate lipid and redox homeostasis in the developing brain. In agreement with this essential role, deficiency of Obp44a leads to anatomical and behavioral deficits in adult animals and elevated oxidized lipid levels. Collectively, our findings unveil the crucial involvement of a noncanonical lipid chaperone to shuttle fatty acids within and outside the brain, as needed to maintain a healthy brain lipid environment. These findings could inspire the design of novel approaches to restore lipid homeostasis that is dysregulated in CNS diseases.
RESUMEN
Melanin pigments are found in most life forms, where they are responsible for coloration and ultraviolet (UV) light protection. Natural melanin is a poorly soluble and complex biosynthesis product produced through confined and templated enzymatic oxidation of tyrosine. It has been challenging to create water-soluble synthetic mimics. This study demonstrates the enzymatic synthesis of oxidized phenols confined inside liquid droplets. We use an amphiphilic, bifunctional peptide, DYFR9, that combines a tyrosine tripeptide previously shown to undergo enzymatic oxidation to form peptide pigments with broad absorbance, and polyarginine to facilitate complex coacervation in the presence of ATP. When ATP, DYFR9 are mixed and exposed to tyrosinase, pigmented liquid droplets result, while no appreciable oxidation is observed in the bulk.
Asunto(s)
Melaninas , Tirosina , Monofenol Monooxigenasa , Péptidos , Adenosina TrifosfatoRESUMEN
Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and buriedness. We demonstrate that our HDX rate data obtained in solution adds value to predictions of dynamics derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site vs. allosteric small-molecule inhibitors. These maps reveal distinct, dramatic, and widespread effects on protein dynamics relative to the apo form, including changes to dynamics in locations distal (>35 Å) from the respective ligand binding sites. These results help shed light on the allosteric nature of PTP1B and the surprisingly far-reaching consequences of inhibitor binding in this important protein. Overall, our work showcases the potential of HDX-MS for elucidating protein conformational dynamics and allosteric effects of small-molecule ligands, and highlights the potential of integrating HDX-MS alongside other complementary methods to guide the development of new therapeutics.
RESUMEN
Major obstacles in brain cancer treatment include the blood-tumor barrier (BTB), which limits the access of most therapeutic agents, and quiescent tumor cells, which resist conventional chemotherapy. Here, we show that Sox2+ tumor cells project cellular processes to ensheathe capillaries in mouse medulloblastoma (MB), a process that depends on the mechanosensitive ion channel Piezo2. MB develops a tissue stiffness gradient as a function of distance to capillaries. Sox2+ tumor cells perceive substrate stiffness to sustain local intracellular calcium, actomyosin tension, and adhesion to promote cellular process growth and cell surface sequestration of ß-catenin. Piezo2 knockout reverses WNT/ß-catenin signaling states between Sox2+ tumor cells and endothelial cells, compromises the BTB, reduces the quiescence of Sox2+ tumor cells, and markedly enhances the MB response to chemotherapy. Our study reveals that mechanosensitive tumor cells construct the BTB to mask tumor chemosensitivity. Targeting Piezo2 addresses the BTB and tumor quiescence properties that underlie treatment failures in brain cancer.
Asunto(s)
Neoplasias Encefálicas , beta Catenina , Ratones , Animales , beta Catenina/metabolismo , beta Catenina/uso terapéutico , Células Endoteliales/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Encéfalo/metabolismo , Canales Iónicos/metabolismo , Barrera Hematoencefálica/metabolismoRESUMEN
Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.
Asunto(s)
Drosophila , Lípidos , Animales , Encéfalo/diagnóstico por imagen , Rayos Láser , Lípidos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Background: Pancreatic beta cells regulate bioenergetics efficiency and secret insulin in response to glucose and nutrient availability. The mechanistic Target of Rapamycin (mTOR) network orchestrates pancreatic progenitor cell growth and metabolism by nucleating two complexes, mTORC1 and mTORC2. Objective: To determine the impact of mTORC1/mTORC2 inhibition on amino acid metabolism in mouse pancreatic beta cells (Beta-TC-6 cells, ATCC-CRL-11506) using high-resolution metabolomics (HRM) and live-mitochondrial functions. Methods: Pancreatic beta TC-6 cells were incubated for 24 h with either: RapaLink-1 (RL); Torin-2 (T); rapamycin (R); metformin (M); a combination of RapaLink-1 and metformin (RLM); Torin-2 and metformin (TM); compared to the control. We applied high-resolution mass spectrometry (HRMS) LC-MS/MS untargeted metabolomics to compare the twenty natural amino acid profiles to the control. In addition, we quantified the bioenergetics dynamics and cellular metabolism by live-cell imaging and the MitoStress Test XF24 (Agilent, Seahorse). The real-time, live-cell approach simultaneously measures the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) to determine cellular respiration and metabolism. Statistical significance was assessed using ANOVA on Ranks and post-hoc Welch t-Tests. Results: RapaLink-1, Torin-2, and rapamycin decreased L-aspartate levels compared to the control (p = 0.006). Metformin alone did not affect L-aspartate levels. However, L-asparagine levels decreased with all treatment groups compared to the control (p = 0.03). On the contrary, L-glutamate and glycine levels were reduced only by mTORC1/mTORC2 inhibitors RapaLink-1 and Torin-2, but not by rapamycin or metformin. The metabolic activity network model predicted that L-aspartate and AMP interact within the same activity network. Live-cell bioenergetics revealed that ATP production was significantly reduced in RapaLink-1 (122.23 ± 33.19), Torin-2 (72.37 ± 17.33) treated cells, compared to rapamycin (250.45 ± 9.41) and the vehicle control (274.23 ± 38.17), p < 0.01. However, non-mitochondrial oxygen consumption was not statistically different between RapaLink-1 (67.17 ± 3.52), Torin-2 (55.93 ± 8.76), or rapamycin (80.01 ± 4.36, p = 0.006). Conclusions: Dual mTORC1/mTORC2 inhibition by RapaLink-1 and Torin-2 differentially altered the amino acid profile and decreased mitochondrial respiration compared to rapamycin treatment which only blocks the FRB domain on mTOR. Third-generation mTOR inhibitors may alter the mitochondrial dynamics and reveal a bioenergetics profile that could be targeted to reduce mitochondrial stress.
Asunto(s)
Células Secretoras de Insulina , Metformina , Aminoácidos/metabolismo , Animales , Ácido Aspártico/metabolismo , Cromatografía Liquida , Metabolismo Energético , Células Secretoras de Insulina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Metformina/farmacología , Ratones , Oxígeno/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Espectrometría de Masas en TándemRESUMEN
Molecular chaperones have an essential role for the maintenance of a balanced protein homeostasis. Here, we investigate how protein kinases are recruited and loaded to the Hsp90-Cdc37 complex, the first step during Hsp90-mediated chaperoning that leads to enhanced client kinase stability and activation. We show that conformational dynamics of all partners is a critical feature of the underlying loading mechanism. The kinome co-chaperone Cdc37 exists primarily in a dynamic extended conformation but samples a low-populated, well-defined compact structure. Exchange between these two states is maintained in an assembled Hsp90-Cdc37 complex and is necessary for substrate loading. Breathing motions at the N-lobe of a free kinase domain partially expose the kinase segment trapped in the Hsp90 dimer downstream in the cycle. Thus, client dynamics poise for chaperone dependence. Hsp90 is not directly involved during loading, and Cdc37 is assigned the task of sensing clients by stabilizing the preexisting partially unfolded client state.
RESUMEN
Water-responsive (WR) materials that reversibly deform in response to humidity changes show great potential for developing muscle-like actuators for miniature and biomimetic robotics. Here, it is presented that Bacillus (B.) subtilis' peptidoglycan (PG) exhibits WR actuation energy and power densities reaching 72.6 MJ m-3 and 9.1 MW m-3 , respectively, orders of magnitude higher than those of frequently used actuators, such as piezoelectric actuators and dielectric elastomers. PG can deform as much as 27.2% within 110 ms, and its actuation pressure reaches ≈354.6 MPa. Surprisingly, PG exhibits an energy conversion efficiency of ≈66.8%, which can be attributed to its super-viscous nanoconfined water that efficiently translates the movement of water molecules to PG's mechanical deformation. Using PG, WR composites that can be integrated into a range of engineering structures are developed, including a robotic gripper and linear actuators, which illustrate the possibilities of using PG as building blocks for high-efficiency WR actuators.
Asunto(s)
Peptidoglicano , Robótica , Elastómeros/química , Músculos , AguaRESUMEN
MAIN CONCLUSION: Metabolites in Rafflesia-infected and non-infected Tetrastigma were compared which may have applications in Rafflesia propagation. Benzylisoquinoline alkaloids, here reported for the first time in Vitaceae, were abundant in non-infected shoots and may be a form of defense. In Rafflesia-infected shoots, oxylipins, which mediate immune response, were elevated. Endemic to the forests of Southeast Asia, Rafflesia (Rafflesiaceae) is a genus of holoparasitic plants producing the largest flowers in the world, yet completely dependent on its host, the tropical grape vine, Tetrastigma. Rafflesia species are threatened with extinction, making them an iconic symbol of plant conservation. Thus far, propagation has proved challenging, greatly decreasing efficacy of conservation efforts. This study compared the metabolites in the shoots of Rafflesia-infected and non-infected Tetrastigma loheri to examine how Rafflesia infection affects host metabolomics and elucidate the Rafflesia infection process. Results from LC-MS-based untargeted metabolomics analysis showed benzylisoquinoline alkaloids were naturally more abundant in non-infected shoots and are here reported for the first time in the genus Tetrastigma, and in the grape family, Vitaceae. These metabolites have been implicated in plant defense mechanisms and may prevent a Rafflesia infection. In Rafflesia-infected shoots, oxygenated fatty acids, or oxylipins, and a flavonoid, previously shown involved in plant immune response, were significantly elevated. This study provides a preliminary assessment of metabolites that differ between Rafflesia-infected and non-infected Tetrastigma hosts and may have applications in Rafflesia propagation to meet conservation goals.
Asunto(s)
Magnoliopsida , Parásitos , Vitaceae , Animales , Flores , ReproducciónRESUMEN
Mitomycin C, (MC), an antitumor drug, is a DNA alkylating agent currently used in the clinics. Inert in its native form, MC is reduced to reactive mitosenes, which undergo nucleophilic attack by guanine or adenine bases in DNA to form monoadducts as well as interstrand crosslinks (ICLs). Although ICLs are considered the most cytotoxic lesions, the role of each individual adduct in the drug's cytotoxicity is still not fully understood. Synthetic routes have been developed to access modified oligonucleotides containing dG MC-monoadducts and dG-MC-dG ICL at a single position of their base sequences to investigate the biological effects of these adducts. However, until now, oligonucleotides containing monoadducts formed by MC at the adenine base had not been available, thus preventing the examination of the role played by these lesions in the toxicity of MC. Here, we present a route to access these substrates. Structural proof of the adducted oligonucleotides were provided by enzymatic digestion to nucleosides and high-resolution mass spectral analysis. Additionally, parent oligonucleotides containing a dG monoadduct and a dG-MC-dG ICL were also produced. The stability and physical properties of all substrates were compared via CD spectroscopy and UV melting temperature studies. Finally, virtual models were created to explore the conformational space and structural features of these MC-DNA complexes.
Asunto(s)
Aductos de ADN , Mitomicina , Adenina , Guanina , OligonucleótidosRESUMEN
Lipid shuttling between neurons and glia contributes to the development, function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, we perform combined genetic, transcriptome, and biochemical analyses in the developing Drosophila larval brain. Here we report, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, we elucidate molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment.
Asunto(s)
Apolipoproteínas/metabolismo , Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Animales Modificados Genéticamente , Apolipoproteínas/genética , Transporte Biológico , Encéfalo/citología , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Perfilación de la Expresión Génica , Humanos , Larva/genética , Larva/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Unión Proteica , Receptores Citoplasmáticos y Nucleares/genéticaRESUMEN
The calmodulin (CaM) activated α-kinase, eukaryotic elongation factor 2 kinase (eEF-2K), plays a central role in regulating translational elongation by phosphorylating eukaryotic elongation factor 2 (eEF-2), thereby reducing its ability to associate with the ribosome and suppressing global protein synthesis. Using TR (for truncated), a minimal functional construct of eEF-2K, and utilizing hydrogen/deuterium exchange mass spectrometry (HXMS), solution-state nuclear magnetic resonance (NMR) and biochemical approaches, we investigate the conformational changes accompanying complex formation between Ca2+ -CaM and TR and the effects of autophosphorylation of the latter at Thr348, its primary regulatory site. Our results suggest that a CaM C-lobe surface, complementary to the one involved in recognizing the calmodulin-binding domain (CBD) of TR, provides a secondary TR-interaction platform. CaM helix F, which is part of this secondary surface, responds to both Thr348 phosphorylation and pH changes, indicating its integration into an allosteric network that encompasses both components of the Ca2+ -CaMâ¢TR complex. Solution NMR data suggest that CaMH107K , which carries a helix F mutation, is compromised in its ability to drive the conformational changes in TR necessary to enable efficient Thr348 phosphorylation. Biochemical studies confirm the diminished capacity of CaMH107K to induce TR autophosphorylation compared to wild-type CaM.
Asunto(s)
Calmodulina/química , Quinasa del Factor 2 de Elongación/química , Resonancia Magnética Nuclear Biomolecular , Sustitución de Aminoácidos , Calmodulina/genética , Quinasa del Factor 2 de Elongación/genética , Humanos , Mutación Missense , Fosforilación , Estructura Cuaternaria de Proteína , Estructura Secundaria de ProteínaRESUMEN
Mitomycinâ C (MC) an antitumor drug and decarbamoylmitomycinâ C (DMC), a derivative of MC lacking the carbamoyl moiety, are DNA alkylating agents which can form DNA interstrand crosslinks (ICLs) between deoxyguanosine residues located on opposing DNA strands. MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α, trans) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-ß, cis). The crosslinking reaction is diastereospecific: trans-crosslinks are formed exclusively at CpG sequences, while cis-crosslinks are formed only at GpC sequences. Until now, oligonucleotides containing 1"-ß-deoxyguanosine adducts or ICL at a specific site could not be synthesized, thus limiting the investigation of the role played by the stereochemical configuration at C1'' in the toxicity of these compounds. Here, a novel biomimetic synthesis to access these substrates is presented. Structural proof of the adducted oligonucleotides and ICL were provided by enzymatic digestion to nucleosides, high resolution mass spectral analysis, CD spectroscopy and UV melting temperature studies. Finally, a virtual model of the 25-mer 1"-ß ICL synthesized was created to explore the conformational space and structural features of the crosslinked duplex.
Asunto(s)
Aductos de ADN , Mitomicinas/química , Oligonucleótidos , ADN/química , Daño del ADN , Oligonucleótidos/síntesis química , Oligonucleótidos/químicaRESUMEN
Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.
Asunto(s)
Métodos Analíticos de la Preparación de la Muestra , Metabolómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Animales , Crioultramicrotomía , Humanos , Ratones Endogámicos C57BLRESUMEN
Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light-sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.
Asunto(s)
Histidina Quinasa/metabolismo , Luz , Adenosina Difosfato/metabolismo , Oscuridad , Activación Enzimática/efectos de la radiación , Histidina Quinasa/química , Modelos Moleculares , Mutación/genética , Dominios Proteicos , Estabilidad Proteica , Estructura Secundaria de ProteínaRESUMEN
Factor Xa (fXa) inhibition by antithrombin (AT) enabled by heparin or heparan sulfate is critical for controlling blood coagulation. AT activation by heparin has been investigated extensively, while interaction of heparin with trapped AT/fXa intermediates has received relatively little attention. We use native electrospray ionization mass spectrometry to study the role of heparin chains of varying length [hexa-, octa-, deca-, and eicosasaccharides (dp6, dp8, dp10, and dp20, respectively)] in AT/fXa complex assembly. Despite being critical promoters of AT/Xa binding, shorter heparin chains are excluded from the final products (trapped intermediates). However, replacement of short heparin segments with dp20 gives rise to a prominent ionic signal of ternary complexes. These species are also observed when the trapped intermediate is initially prepared in the presence of a short oligoheparin (dp6), followed by addition of a longer heparin chain (dp20), indicating that binding of heparin to AT/fXa complexes takes place after the inhibition event. The importance of the heparin chain length for its ability to associate with the trapped intermediate suggests that the binding likely occurs in a bidentate fashion (where two distinct segments of oligoheparin make contacts with the protein components, while the part of the chain separating these two segments is extended into solution to minimize electrostatic repulsion). This model is corroborated by both molecular dynamics simulations with an explicit solvent and ion mobility measurements in the gas phase. The observed post-inhibition binding of heparin to the trapped AT/fXa intermediates hints at the likely role played by heparan sulfate in their catabolism.
Asunto(s)
Antitrombinas/química , Factor Xa/química , Glicosaminoglicanos/química , Coagulación Sanguínea , Cromatografía en Gel , Heparina/química , Humanos , Espectrometría de MasasRESUMEN
Eukaryotic elongation factor 2 kinase (eEF-2K), the only known calmodulin (CaM)-activated α-kinase, phosphorylates eukaryotic elongation factor 2 (eEF-2) on a specific threonine (Thr-56) diminishing its affinity for the ribosome and reducing the rate of nascent chain elongation during translation. Despite its critical cellular role, the precise mechanisms underlying the CaM-mediated activation of eEF-2K remain poorly defined. Here, employing a minimal eEF-2K construct (TR) that exhibits activity comparable to the wild-type enzyme and is fully activated by CaM in vitro and in cells, and using a variety of complimentary biophysical techniques in combination with computational modeling, we provide a structural mechanism by which CaM activates eEF-2K. Native mass analysis reveals that CaM, with two bound Ca2+ ions, forms a stoichiometric 1:1 complex with TR. Chemical crosslinking mass spectrometry and small-angle X-ray scattering measurements localize CaM near the N-lobe of the TR kinase domain and the spatially proximal C-terminal helical repeat. Hydrogen/deuterium exchange mass spectrometry and methyl NMR indicate that the conformational changes induced on TR by the engagement of CaM are not localized but are transmitted to remote regions that include the catalytic site and the functionally important phosphate binding pocket. The structural insights obtained from the present analyses, together with our previously published kinetics data, suggest that TR, and by inference, wild-type eEF-2K, upon engaging CaM undergoes a conformational transition resulting in a state that is primed to efficiently auto-phosphorylate on the primary activating T348 en route to full activation.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Quinasa del Factor 2 de Elongación/química , Quinasa del Factor 2 de Elongación/metabolismo , Calmodulina/química , Calmodulina/genética , Quinasa del Factor 2 de Elongación/genética , Humanos , Cinética , Fosforilación , Conformación ProteicaRESUMEN
Melanins are a family of heterogeneous polymeric pigments that provide ultraviolet (UV) light protection, structural support, coloration, and free radical scavenging. Formed by oxidative oligomerization of catecholic small molecules, the physical properties of melanins are influenced by covalent and noncovalent disorder. We report the use of tyrosine-containing tripeptides as tunable precursors for polymeric pigments. In these structures, phenols are presented in a (supra-)molecular context dictated by the positions of the amino acids in the peptide sequence. Oxidative polymerization can be tuned in a sequence-dependent manner, resulting in peptide sequence-encoded properties such as UV absorbance, morphology, coloration, and electrochemical properties over a considerable range. Short peptides have low barriers to application and can be easily scaled, suggesting near-term applications in cosmetics and biomedicine.