RESUMO
Toll-like receptors (TLRs) are key players in the innate immune system. Despite the great efforts in TLR structural biology, today we know the spatial structures of only four human TLR intracellular TIR domains. All of them belong to one of five subfamilies of receptors. One of the main bottlenecks is the high-level production of correctly folded proteins in soluble form. Here we used a rational approach to find the optimal parameters to produce TIR domains of all ten human TLR family members in soluble form in E. coli cells. We showed that dozens of milligrams of soluble His-tagged TLR2/3/6/7TIR and MBP-tagged TLR3/5/7/8TIR can be produced. We also developed the purification protocols and demonstrated by CD and NMR spectroscopy that purified TLR2/3/7TIR demonstrate a structural organization inherent to TIR domains. This illustrates the correct folding of produced proteins and their suitability for further structural and functional investigations.
Assuntos
Domínios Proteicos , Receptores Toll-Like , Humanos , Receptores Toll-Like/metabolismo , Receptores Toll-Like/química , Escherichia coli/metabolismo , Escherichia coli/genética , Dobramento de ProteínaRESUMO
In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.
Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Animais , Fluorescência , MutaçãoRESUMO
Emfourin (M4in) is a protein metalloprotease inhibitor recently discovered in the bacterium Serratia proteamaculans and the prototype of a new family of protein protease inhibitors with an unknown mechanism of action. Protealysin-like proteases (PLPs) of the thermolysin family are natural targets of emfourin-like inhibitors widespread in bacteria and known in archaea. The available data indicate the involvement of PLPs in interbacterial interaction as well as bacterial interaction with other organisms and likely in pathogenesis. Arguably, emfourin-like inhibitors participate in the regulation of bacterial pathogenesis by controlling PLP activity. Here, we determined the 3D structure of M4in using solution NMR spectroscopy. The obtained structure demonstrated no significant similarity to known protein structures. This structure was used to model the M4in-enzyme complex and the complex model was verified by small-angle X-ray scattering. Based on the model analysis, we propose a molecular mechanism for the inhibitor, which was confirmed by site-directed mutagenesis. We show that two spatially close flexible loop regions are critical for the inhibitor-protease interaction. One region includes aspartic acid forming a coordination bond with catalytic Zn2+ of the enzyme and the second region carries hydrophobic amino acids interacting with protease substrate binding sites. Such an active site structure corresponds to the noncanonical inhibition mechanism. This is the first demonstration of such a mechanism for protein inhibitors of thermolysin family metalloproteases, which puts forward M4in as a new basis for the development of antibacterial agents relying on selective inhibition of prominent factors of bacterial pathogenesis belonging to this family.
Assuntos
Proteínas de Bactérias , Metaloproteases , Termolisina/metabolismo , Proteínas de Bactérias/metabolismo , Metaloproteases/genética , Espectroscopia de Ressonância Magnética , Peptídeo HidrolasesRESUMO
Human InsR, IGF1R, and IRR receptor tyrosine kinases (RTK) of the insulin receptor subfamily play an important role in signaling pathways for a wide range of physiological processes and are directly associated with many pathologies, including neurodegenerative diseases. The disulfide-linked dimeric structure of these receptors is unique among RTKs. Sharing high sequence and structure homology, the receptors differ dramatically in their localization, expression, and functions. In this work, using high-resolution NMR spectroscopy supported by atomistic computer modeling, conformational variability of the transmembrane domains and their interactions with surrounding lipids were found to differ significantly between representatives of the subfamily. Therefore, we suggest that the heterogeneous and highly dynamic membrane environment should be taken into account in the observed diversity of the structural/dynamic organization and mechanisms of activation of InsR, IGF1R, and IRR receptors. This membrane-mediated control of receptor signaling offers an attractive prospect for the development of new targeted therapies for diseases associated with dysfunction of insulin subfamily receptors.
Assuntos
Desenvolvimento de Medicamentos , Receptor de Insulina , Humanos , Domínios Proteicos , Receptor de Insulina/química , Receptor de Insulina/fisiologia , Transdução de SinaisRESUMO
Membrane proteins are one of the keystone objects in molecular biology, but their structural studies often require an extensive search for an appropriate membrane-like environment and an efficient refolding protocol for a recombinant protein. Isotropic bicelles are a convenient membrane mimetic used in structural studies of membrane proteins. Helical membrane domains are often transferred into bicelles from trifluoroethanol-water mixtures. However, the protocols for such a refolding are empirical and the process itself is still not understood in detail. In search of the optimal refolding approaches for helical membrane proteins, we studied here how membrane proteins, lipids, and detergents interact with each other at various trifluoroethanol-water ratios. Using high-resolution NMR spectroscopy and dynamic light scattering, we determined the key states of the listed compounds in the trifluoroethanol/water mixture, found the factors that could be critical for the efficiency of refolding, and proposed several most optimal protocols. These protocols were developed on the transmembrane domain of neurotrophin receptor TrkA and tested on two model helical membrane domains-transmembrane of Toll-like receptor TLR9 and voltage-sensing domain of a potassium channel KvAP.
Assuntos
Trifluoretanol , Água , Ressonância Magnética Nuclear Biomolecular , Proteínas de Membrana , Lipídeos/químicaRESUMO
Apamin is often cited as one of the few substances selectively acting on small-conductance Ca2+-activated potassium channels (KCa2). However, published pharmacological and structural data remain controversial. Here, we investigated the molecular pharmacology of apamin by two-electrode voltage-clamp in Xenopus laevis oocytes and patch-clamp in HEK293, COS7, and CHO cells expressing the studied ion channels, as well as in isolated rat brain neurons. The microtitre broth dilution method was used for antimicrobial activity screening. The spatial structure of apamin in aqueous solution was determined by NMR spectroscopy. We tested apamin against 42 ion channels (KCa, KV, NaV, nAChR, ASIC, and others) and confirmed its unique selectivity to KCa2 channels. No antimicrobial activity was detected for apamin against Gram-positive or Gram-negative bacteria. The NMR solution structure of apamin was deposited in the Protein Data Bank. The results presented here demonstrate that apamin is a selective nanomolar or even subnanomolar-affinity KCa2 inhibitor with no significant effects on other molecular targets. The spatial structure as well as ample functional data provided here support the use of apamin as a KCa2-selective pharmacological tool and as a template for drug design.
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NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH-N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.
Assuntos
Rodanina , Ligação de Hidrogênio , Ligantes , Espectroscopia de Ressonância Magnética , ProteínasRESUMO
The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552.
Assuntos
Heme , Tiocianatos , Grupo dos Citocromos c , Ectothiorhodospiraceae , Escherichia coli/metabolismo , Heme/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Oxirredutases/metabolismoRESUMO
"Fluorescence-Activating and absorption-Shifting Tag" (FAST) is a well-studied fluorogen-activating protein with high brightness and low size, able to activate a wide range of fluorogens. This makes FAST a promising target for both protein and fluorogen optimization. Here, we describe the structure-based rational design of the enhanced FAST mutants, optimized for the N871b fluorogen. Using the spatial structure of the FAST/N871b complex, NMR relaxation analysis, and computer simulations, we identify the mobile regions in the complex and suggest mutations that could stabilize both the protein and the ligand. Two of our mutants appear brighter than the wild-type FAST, and these mutants provide up to 35% enhancement for several other fluorogens of similar structure, both in vitro and in vivo. Analysis of the mutants by NMR reveals that brighter mutants demonstrate the highest stability and lowest length of intermolecular H-bonds. Computer simulations provide the structural basis for such stabilization.
Assuntos
Corantes Fluorescentes , Proteínas , Fluorescência , Corantes Fluorescentes/químicaRESUMO
Receptor tyrosine kinases (RTKs) are key players in development and several diseases. Understanding the molecular mechanism of RTK activation by its ligand could lead to the design of new RTK inhibitors. How the extracellular domain is coupled to the intracellular kinase domain is a matter of debate. Ligand-induced dimerization and ligand-induced conformational change of pre-formed dimers are two of the most proposed models. Recently we proposed that TrkA, the RTK for nerve growth factor (NGF), is activated by rotation of the transmembrane domain (TMD) pre-formed dimers upon NGF binding. However, one of the unsolved issues is how the ligand binding is conformationally coupled to the TMD rotation if unstructured extracellular juxtamembrane (eJTM) regions separate them. Here we use nuclear magnetic resonance in bicelles and functional studies to demonstrate that eJTM regions from the Trk family are intrinsically disordered and couple the ligand-binding domains and TMDs possibly via the interaction with NGF.
RESUMO
Thionins are the family of small (â¼5 kDa) cationic cysteine-rich peptides involved in the immune response in plants. Viscotoxin A3 (VtA3) is the thionin from mistletoe (Viscum album) demonstrating antimicrobial and cytotoxic activity against cancer cells in vitro. VtA3 (charge +6) interacts with the membranes containing anionic lipids and forms cation-selective ion channels. Here we studied the VtA3 structure in membrane-mimicking media by NMR spectroscopy. Spatial structure of VtA3, consisting of a helical hairpin and a short ß-sheet, was stable and did not undergo significant changes during micelle binding. VtA3 molecule bound with high affinity to the surface of zwitterionic dodecylphosphocholine (DPC) micelle by hydrophobic patch in the helical hairpin. Oligomerization of VtA3 was observed in the anionic micelles of sodium dodecylsulphate (SDS). No direct contacts between the peptide molecules were observed and the possible interfaces of detergent-assisted oligomerization were revealed. The data obtained suggest that the VtA3 membrane activity, depending on the concentration, obeys the 'toroidal' pore model or the 'carpet' mechanism. The model of the membrane disrupting complex, which explains the ion channel formation in the partially anionic membranes, was proposed.
Assuntos
Membrana Celular/química , Detergentes/química , Canais Iônicos/química , Micelas , Proteínas de Plantas/química , Dodecilsulfato de Sódio/química , Viscum album/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Canais Iônicos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Folhas de Planta/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Caules de Planta/química , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Homologia de Sequência de AminoácidosRESUMO
Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-ß peptide (Aß) oligomers play a crucial role in AD pathogenesis. All-d-Enantiomeric peptide D3 and its derivatives were developed to disassemble and destroy cytotoxic Aß aggregates. One of the D3-like compounds is approaching phase II clinical trials; however, high-resolution details of its disease-preventing or pharmacological actions are not completely clear. We demonstrate that peptide D3 stabilizing Aß monomer dynamically interacts with the extracellular juxtamembrane region of a membrane-bound fragment of an amyloid precursor protein containing the Aß sequence. MD simulations based on NMR measurement results suggest that D3 targets the amyloidogenic region, not compromising its α-helicity and preventing intermolecular hydrogen bonding, thus creating prerequisites for inhibition of early steps of Aß conversion into ß-conformation and its toxic oligomerization. An enhanced understanding of the D3 action molecular mechanism facilitates development of effective AD treatment and prevention strategies.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/metabolismo , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Ligação Proteica , EstereoisomerismoRESUMO
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation.
Assuntos
Receptor 1 Toll-Like/genética , Zinco/metabolismo , Células HEK293 , Humanos , Íons/metabolismo , Domínios Proteicos , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismoRESUMO
The neurotrophin receptors p75 and tyrosine protein kinase receptor A (TrkA) play important roles in the development and survival of the nervous system. Biochemical data suggest that p75 and TrkA reciprocally regulate the activities of each other. For instance, p75 is able to regulate the response of TrkA to lower concentrations of nerve growth factor (NGF), and TrkA promotes shedding of the extracellular domain of p75 by α-secretases in a ligand-dependent manner. The current model suggests that p75 and TrkA are regulated by means of a direct physical interaction; however, the nature of such interaction has been elusive thus far. Here, using NMR in micelles, multiscale molecular dynamics, FRET, and functional studies, we identified and characterized the direct interaction between TrkA and p75 through their respective transmembrane domains (TMDs). Molecular dynamics of p75-TMD mutants suggests that although the interaction between TrkA and p75 TMDs is maintained upon mutation, a specific protein interface is required to facilitate TrkA active homodimerization in the presence of NGF. The same mutations in the TMD protein interface of p75 reduced the activation of TrkA by NGF as well as reducing cell differentiation. In summary, we provide a structural model of the p75-TrkA receptor complex necessary for neuronal development stabilized by TMD interactions.
Assuntos
Receptor de Fator de Crescimento Neural , Receptor trkA , Animais , Diferenciação Celular , Neurogênese , Células PC12 , Ligação Proteica , Domínios Proteicos , RatosRESUMO
Alzheimer's disease is the most common type of neurodegenerative disease in the world. Genetic evidence strongly suggests that aberrant generation, aggregation, and/or clearance of neurotoxic amyloid-ß peptides (Aß) triggers the disease. Aß accumulates at the points of contact of neurons in ordered cords and fibrils, forming the so-called senile plaques. Aß isoforms of different lengths are found in healthy human brains regardless of age and appear to play a role in signaling pathways in the brain and to have neuroprotective properties at low concentrations. In recent years, different substances have been developed targeting Aß production, aggregation, interaction with other molecules, and clearance, including peptide-based drugs. Aß is a product of sequential cleavage of the membrane glycoprotein APP (amyloid precursor protein) by ß- and γ-secretases. A number of familial mutations causing an early onset of the disease have been identified in the APP, especially in its transmembrane domain. The mutations are reported to influence the production, oligomerization, and conformational behavior of Aß peptides. This review highlights the results of structural studies of the main proteins involved in Alzheimer's disease pathogenesis and the molecular mechanisms by which perspective therapeutic substances can affect Aß production and nucleation.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Conformação Proteica , Animais , Humanos , Agregados Proteicos , Mapas de Interação de Proteínas , ProteóliseRESUMO
Isotope-labeled antiviral drug Triazavirin containing 2H, 13C, and 15N atoms in its structure has been synthesized. 13C2H3I and KS13CN served as donors of 13C isotopes. The use of 13С-MeI containing 2H atoms made it possible to additionally incorporate deuterium labels into the structure of the compound. The 15N atoms were incorporated using 15N-enriched sodium nitrite, aminoguanidine carbonate, and ethyl nitroacetate. The resulting 2H3,13C2,15N3-Triazavirin was characterized by NMR spectroscopy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10593-021-02927-1.
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One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.
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Old world scorpions produce an abundance of toxins called α-NaTx, which interfere with the fast inactivation of voltage-gated sodium channels. Their selectivity to channels of mammals or insects depends on a part of toxin named the specificity module. We report here the spatial structure of a major and broadly active toxin MeuNaTxα-1 from the venom of Mesobuthus eupeus. Notably, its specificity module is markedly different from other α-NaTx with known 3D structure. Close inspection shows that its conformation is a result of an interplay between protein motifs such as the nest and niche, which eventually shape α-NaTx structural diversity.
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T7 expression system is an extremely popular approach for the recombinant protein production in Escherichia coli for structural and functional studies and therapeutic applications. There are many useful tools and successful techniques that allow expressing the desired protein in this system. However, high yield of soluble protein often requires a systematic optimization of a wide range of cell cultivation parameters. Here we analyze the effect of three key cultivation parameters - chemical inductor, temperature and time of post-induction culturing on the expression level of TLR1 intracellular TIR domain in a soluble form. In addition, the influence of Triton X-100 detergent on the protein solubility during the cell lysis was investigated. We show that a high expression level of the correctly folded soluble protein can be obtained under different combinations of cultivation parameters.
Assuntos
Escherichia coli , Expressão Gênica , Receptor 1 Toll-Like , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Receptor 1 Toll-Like/biossíntese , Receptor 1 Toll-Like/genéticaRESUMO
Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three ß-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.