RESUMO
Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that ß1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. ß1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and ß1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that ß1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, ß1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.This article has an associated First Person interview with the first author of the paper.
Assuntos
Aorta/fisiologia , Integrina beta1/metabolismo , Fluxo Sanguíneo Regional/fisiologia , Animais , Linhagem Celular , Feminino , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina beta1/genética , Mecanorreceptores/fisiologia , Camundongos , Camundongos Knockout , Estresse Fisiológico/fisiologiaRESUMO
Human flap endonuclease-1 (hFEN1) catalyzes the divalent metal ion-dependent removal of single-stranded DNA protrusions known as flaps during DNA replication and repair. Substrate selectivity involves passage of the 5'-terminus/flap through the arch and recognition of a single nucleotide 3'-flap by the α2-α3 loop. Using NMR spectroscopy, we show that the solution conformation of free and DNA-bound hFEN1 are consistent with crystal structures; however, parts of the arch region and α2-α3 loop are disordered without substrate. Disorder within the arch explains how 5'-flaps can pass under it. NMR and single-molecule FRET data show a shift in the conformational ensemble in the arch and loop region upon addition of DNA. Furthermore, the addition of divalent metal ions to the active site of the hFEN1-DNA substrate complex demonstrates that active site changes are propagated via DNA-mediated allostery to regions key to substrate differentiation. The hFEN1-DNA complex also shows evidence of millisecond timescale motions in the arch region that may be required for DNA to enter the active site. Thus, hFEN1 regional conformational flexibility spanning a range of dynamic timescales is crucial to reach the catalytically relevant ensemble.
Assuntos
Endonucleases Flap/química , Domínio Catalítico , Cátions Bivalentes/química , DNA/química , DNA/metabolismo , Endonucleases Flap/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfatos/química , Conformação Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Myelocytomatosis proto-oncogene transcription factor (Myc) is an intrinsically disordered protein with critical roles in cellular homeostasis and neoplastic transformation. It is tightly regulated in the cell, with Myc phosphorylation playing a major role. In addition to the well-described tandem phosphorylation of Thr-52 and Ser-62 in the Myc transactivation domain linked to its degradation, P21 (RAC1)-activated kinase 2 (PAK2)-mediated phosphorylation of serine and threonine residues in the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) region regulates Myc transcriptional activity. Here we report that PAK2 preferentially phosphorylates Myc twice, at Thr-358 and Ser-373, with only a minor fraction being modified at the previously identified Thr-400 site. For transcriptional activity, Myc binds E-box DNA elements, requiring its heterodimerization with Myc-associated factor X (Max) via the bHLH-LZ regions. Using isothermal calorimetry (ITC), we found that Myc phosphorylation destabilizes this ternary protein-DNA complex by decreasing Myc's affinity for Max by 2 orders of magnitude, suggesting a major effect of phosphorylation on this complex. Phosphomimetic substitutions revealed that Ser-373 dominates the effect on Myc-Max heterodimerization. Moreover, a T400D substitution disrupted Myc's affinity for Max. ITC, NMR, and CD analyses of several Myc variants suggested that the effect of phosphorylation on the Myc-Max interaction is caused by secondary structure disruption during heterodimerization rather than by a change in the structurally disordered state of Myc or by phosphorylation-induced electrostatic repulsion in the heterodimer. Our findings provide critical insights into the effects of PAK2-catalyzed phosphorylation of Myc on its interactions with Max and DNA.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , DNA/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Mapas de Interação de Proteínas , Estabilidade Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/químicaRESUMO
ß-Phosphoglucomutase (ßPGM) catalyzes isomerization of ß-D-glucose 1-phosphate (ßG1P) into D-glucose 6-phosphate (G6P) via sequential phosphoryl transfer steps using a ß-D-glucose 1,6-bisphosphate (ßG16BP) intermediate. Synthetic fluoromethylenephosphonate and methylenephosphonate analogs of ßG1P deliver novel step 1 transition state analog (TSA) complexes for ßPGM, incorporating trifluoromagnesate and tetrafluoroaluminate surrogates of the phosphoryl group. Within an invariant protein conformation, the ß-D-glucopyranose ring in the ßG1P TSA complexes (step 1) is flipped over and shifted relative to the G6P TSA complexes (step 2). Its equatorial hydroxyl groups are hydrogen-bonded directly to the enzyme rather than indirectly via water molecules as in step 2. The (C)O-P bond orientation for binding the phosphate in the inert phosphate site differs by â¼ 30° between steps 1 and 2. By contrast, the orientations for the axial O-Mg-O alignment for the TSA of the phosphoryl group in the catalytic site differ by only â¼ 5°, and the atoms representing the five phosphorus-bonded oxygens in the two transition states (TSs) are virtually superimposable. The conformation of ßG16BP in step 1 does not fit into the same invariant active site for step 2 by simple positional interchange of the phosphates: the TS alignment is achieved by conformational change of the hexose rather than the protein.
Assuntos
Hexoses/química , Hexoses/metabolismo , Organofosfonatos/química , Organofosfonatos/metabolismo , Fosfoglucomutase/química , Fosfoglucomutase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Catálise , Cristalografia por Raios X , Flúor/química , Glucose-6-Fosfato/química , Glucose-6-Fosfato/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Isomerismo , Cinética , Lactococcus lactis/enzimologia , Magnésio/química , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , TermodinâmicaRESUMO
The 1994 structure of a transition-state analogue with AlF4- and GDP complexed to G1α, a small Gâ protein, heralded a new field of research into the structure and mechanism of enzymes that manipulate the transfer of phosphoryl (PO3- ) groups. The number of enzyme structures in the PDB containing metal fluorides (MFx ) as ligands that imitate either a phosphoryl or a phosphate group was 357 at the end of 2016. They fall into three distinct geometrical classes: 1)â Tetrahedral complexes based on BeF3- that mimic ground-state phosphates; 2)â octahedral complexes, primarily based on AlF4- , which mimic "in-line" anionic transition states for phosphoryl transfer; and 3)â trigonal bipyramidal complexes, represented by MgF3- and putative AlF30 moieties, which mimic the geometry of the transition state. The interpretation of these structures provides a deeper mechanistic understanding into the behavior and manipulation of phosphate monoesters in molecular biology. This Review provides a comprehensive overview of these structures, their uses, and their computational development.
Assuntos
Compostos de Alumínio/química , Fluoretos/química , Fosfatos/metabolismo , Fosfoglicerato Quinase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Compostos de Alumínio/metabolismo , Animais , Fluoretos/metabolismo , Humanos , Modelos Moleculares , Fosfatos/químicaRESUMO
We report X-ray crystallographic and 19 Fâ NMR studies of the G-protein RhoA complexed with MgF3- , GDP, and RhoGAP, which has the mutation Arg85'Ala. When combined with DFT calculations, these data permit the identification of changes in transition state (TS) properties. The X-ray data show how Tyr34 maintains solvent exclusion and the core H-bond network in the active site by relocating to replace the missing Arg85' sidechain. The 19 Fâ NMR data show deshielding effects that indicate the main function of Arg85' is electronic polarization of the transferring phosphoryl group, primarily mediated by H-bonding to O3G and thence to PG . DFT calculations identify electron-density redistribution and pinpoint why the TS for guanosine 5'-triphosphate (GTP) hydrolysis is higher in energy when RhoA is complexed with RhoGAPArg85'Ala relative to wild-type (WT) RhoGAP. This study demonstrates that 19 Fâ NMR measurements, in combination with X-ray crystallography and DFT calculations, can reliably dissect the response of small GTPases to site-specific modifications.
Assuntos
Teoria da Densidade Funcional , GTP Fosfo-Hidrolases/genética , Cristalografia por Raios X , Flúor/química , GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , MutaçãoRESUMO
Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use (19)Fâ NMR chemical shifts in the MgF3(-) transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ-PO3(-) oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ-PO3(-) group based on the structure of a RhoA/GAP-GDP-MgF3(-) TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85' side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln-63 carboxamide by Arg85' to preclude direct hydrogen bonding from water to the target γ-PO3(-) group.
Assuntos
Flúor/metabolismo , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Proteína rhoA de Ligação ao GTP/metabolismo , HidróliseRESUMO
The self-association of prion protein (PrP) is a critical step in the pathology of prion diseases. It is increasingly recognized that small non-fibrillar ß-sheet-rich oligomers of PrP may be of crucial importance in the prion disease process. Here, we characterize the structure of a well defined ß-sheet-rich oligomer, containing â¼12 PrP molecules, and often enclosing a central cavity, formed using full-length recombinant PrP. The N-terminal region of prion protein (residues 23-90) is required for the formation of this distinct oligomer; a truncated form comprising residues 91-231 forms a broad distribution of aggregated species. No infectivity or toxicity was found using cell and animal model systems. This study demonstrates that examination of the full repertoire of conformers and assembly states that can be accessed by PrP under specific experimental conditions should ideally be done using the full-length protein.
Assuntos
Amiloide/química , Doenças Priônicas/metabolismo , Príons/química , Estrutura Terciária de Proteína , Amiloide/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Doenças Priônicas/patologia , Príons/metabolismo , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/químicaRESUMO
Spectral resolution in proton NMR spectroscopy is reduced by the splitting of resonances into multiplets due to the effect of homonuclear scalar couplings. Although these effects are often hidden in protein NMR spectroscopy by low digital resolution and routine apodization, behind the scenes homonuclear scalar couplings increase spectral overcrowding. The possibilities for biomolecular NMR offered by new pure shift NMR methods are illustrated here. Both resolution and sensitivity are improved, without any increase in experiment time. In these experiments, free induction decays are collected in short bursts of data acquisition, with durations short on the timescale of J-evolution, interspersed with suitable refocusing elements. The net effect is real-time (t 2) broadband homodecoupling, suppressing the multiplet structure caused by proton-proton interactions. The key feature of the refocusing elements is that they discriminate between the resonances of active (observed) and passive (coupling partner) spins. This can be achieved either by using band-selective refocusing or by the BIRD element, in both cases accompanied by a nonselective 180° proton pulse. The latter method selects the active spins based on their one-bond heteronuclear J-coupling to (15)N, while the former selects a region of the (1)H spectrum. Several novel pure shift experiments are presented, and the improvements in resolution and sensitivity they provide are evaluated for representative samples: the N-terminal domain of PGK; ubiquitin; and two mutants of the small antifungal protein PAF. These new experiments, delivering improved sensitivity and resolution, have the potential to replace the current standard HSQC experiments.
Assuntos
Proteínas Fúngicas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Penicillium chrysogenum/metabolismo , Ubiquitina/química , Antifúngicos/química , Mutação , Isótopos de Nitrogênio/química , Fosfoglicerato Quinase/química , Dobramento de Proteína , Prótons , Sensibilidade e EspecificidadeRESUMO
Although protein folding is often described by motion on a funnel-shaped overall topology of the energy landscape, the many local interactions that can occur result in considerable landscape roughness which slows folding by increasing internal friction. Recent experimental results have brought to light that this roughness also causes unusual diffusional behaviour of the backbone of an unfolded protein, i.e. the relative motion of protein sections cannot be described by the normal diffusion equation, but shows strongly subdiffusional behaviour with a nonlinear time dependence of the mean square displacement, ãr(2)(t)ãât(α) (α⪠1). This results in significantly slower configurational equilibration than had been assumed hitherto. Analysis of the results also allows quantification of the energy landscape roughness, i.e. the root-mean-squared depth of local minima, yielding a value of 4-5kBT for a typical small protein.
Assuntos
Peptídeos/química , Proteínas/química , Sequência de Aminoácidos , Difusão , Dados de Sequência Molecular , Movimento , Peptídeos/metabolismo , Dobramento de Proteína , Proteínas/metabolismo , TermodinâmicaRESUMO
The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k(B)T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k(B)T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.
Assuntos
Dobramento de Proteína , Proteínas/química , Modelos MolecularesRESUMO
Experimental observations of fluoromagnesate and fluoroaluminate complexes of ß-phosphoglucomutase (ß-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of ß-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of ß-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.
Assuntos
Fosfotransferases (Fosfomutases)/química , Fosfotransferases (Fosfomutases)/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Berílio/química , Cristalografia por Raios X , Fluoretos/química , Lactococcus lactis/enzimologia , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eletricidade Estática , TermodinâmicaRESUMO
Enzymes facilitating the transfer of phosphate groups constitute the most extensive protein families across all kingdoms of life. They make up approximately 10% of the proteins found in the human genome. Understanding the mechanisms by which enzymes catalyze these reactions is essential in characterizing the processes they regulate. Metal fluorides can be used as multifunctional tools to study these enzymes. These ionic species bear the same charge as phosphate and the transferring phosphoryl group and, in addition, allow the enzyme to be trapped in catalytically important states with spectroscopically sensitive atoms interacting directly with active site residues. The ionic nature of these phosphate surrogates also allows their removal and replacement with other analogs. Here, we describe the best practices to obtain these complexes, their use in NMR, X-ray crystallography, cryo-EM, and SAXS and describe a new metal fluoride, scandium tetrafluoride, which has significant anomalous signal using soft X-rays.
RESUMO
Metabolic regulation occurs through precise control of enzyme activity. Allomorphy is a post-translational fine control mechanism where the catalytic rate is governed by a conformational switch that shifts the enzyme population between forms with different activities. ß-Phosphoglucomutase (ßPGM) uses allomorphy in the catalysis of isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Herein, we describe structural and biophysical approaches to reveal its allomorphic regulatory mechanism. Binding of the full allomorphic activator ß-glucose 1,6-bisphosphate stimulates enzyme closure, progressing through NAC I and NAC III conformers. Prior to phosphoryl transfer, loops positioned on the cap and core domains are brought into close proximity, modulating the environment of a key proline residue. Hence accelerated isomerisation, likely via a twisted anti/C4-endo transition state, leads to the rapid predominance of active cis-P ßPGM. In contrast, binding of the partial allomorphic activator fructose 1,6-bisphosphate arrests ßPGM at a NAC I conformation and phosphoryl transfer to both cis-P ßPGM and trans-P ßPGM occurs slowly. Thus, allomorphy allows a rapid response to changes in food supply while not otherwise impacting substantially on levels of important metabolites.
Assuntos
Domínio Catalítico , Fosfoglucomutase , Prolina , Fosfoglucomutase/metabolismo , Fosfoglucomutase/química , Fosfoglucomutase/genética , Prolina/metabolismo , Prolina/química , Isomerismo , Glucofosfatos/metabolismo , Conformação Proteica , Humanos , Catálise , Modelos Moleculares , Glucose-6-Fosfato/análogos & derivadosRESUMO
Arginine phosphorylation plays numerous roles throughout biology. Arginine kinase (AK) catalyzes the delivery of an anionic phosphoryl group (PO3-) from ATP to a planar, trigonal nitrogen in a guanidinium cation. Density functional theory (DFT) calculations have yielded a model of the transition state (TS) for the AK-catalyzed reaction. They reveal a network of over 50 hydrogen bonds that delivers unprecedented pyramidalization and out-of-plane polarization of the arginine guanidinium nitrogen (Nη2) and aligns the electron density on Nη2 with the scissile P-O bond, leading to in-line phosphoryl transfer via an associative mechanism. In the reverse reaction, the hydrogen-bonding network enforces the conformational distortion of a bound phosphoarginine substrate to increase the basicity of Nη2. This enables Nη2 protonation, which triggers PO3- migration to generate ATP. This polarization-pyramidalization of nitrogen in the arginine side chain is likely a general phenomenon that is exploited by many classes of enzymes mediating the post-translational modification of arginine.
RESUMO
The backbone 1H, 13C and 15N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1H-15N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2H, 13C, 15N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography.
Assuntos
Domínio Catalítico , Ressonância Magnética Nuclear Biomolecular , Humanos , Ligantes , Isótopos de NitrogênioRESUMO
Coupling of fast protein dynamics to enzyme chemistry is controversial and has ignited considerable debate, especially over the past 15 years in relation to enzyme-catalyzed H-transfer. H-transfer can occur by quantum tunneling, and the temperature dependence of kinetic isotope effects (KIEs) has emerged as the "gold standard" descriptor of these reactions. The anomalous temperature dependence of KIEs is often rationalized by invoking fast motions to facilitate H-transfer, yet crucially, direct evidence for coupled motions is lacking. The fast motions hypothesis underpinning the temperature dependence of KIEs is based on inference. Here, we have perturbed vibrational motions in pentaerythritol tetranitrate reductase (PETNR) by isotopic substitution where all non-exchangeable atoms were replaced with the corresponding heavy isotope ((13)C, (15)N, and (2)H). The KIE temperature dependence is perturbed by heavy isotope labeling, demonstrating a direct link between (promoting) vibrations in the protein and the observed KIE. Further we show that temperature-independent KIEs do not necessarily rule out a role for fast dynamics coupled to reaction chemistry. We show causality between fast motions and enzyme chemistry and demonstrate how this impacts on experimental KIEs for enzyme reactions.
Assuntos
Hidrogênio/química , Oxirredutases/química , Vibração , Modelos Moleculares , Temperatura , TermodinâmicaRESUMO
Prior evidence supporting the direct observation of phosphorane intermediates in enzymatic phosphoryl transfer reactions was based on the interpretation of electron density corresponding to trigonal species bridging the donor and acceptor atoms. Close examination of the crystalline state of beta-phosphoglucomutase, the archetypal phosphorane intermediate-containing enzyme, reveals that the trigonal species is not PO-3 , but is MgF-3 (trifluoromagnesate). Although MgF-3 complexes are transition state analogues rather than phosphoryl group transfer reaction intermediates, the presence of fluorine nuclei in near-transition state conformations offers new opportunities to explore the nature of the interactions, in particular the independent measures of local electrostatic and hydrogen-bonding distributions using 19F NMR. Measurements on three beta-PGM-MgF-3 -sugar phosphate complexes show a remarkable relationship between NMR chemical shifts, primary isotope shifts, NOEs, cross hydrogen bond F...H-N scalar couplings, and the atomic positions determined from the high-resolution crystal structure of the beta-PGM-MgF--3 -G6P complex. The measurements provide independent validation of the structural and isoelectronic MgF--3 model of near-transition state conformations.
Assuntos
Fluoretos/química , Compostos de Magnésio/química , Fosfoglucomutase/química , Fosforanos/química , Domínio Catalítico , Cristalografia por Raios X , Fluoretos/metabolismo , Glucofosfatos/química , Glucofosfatos/metabolismo , Ligação de Hidrogênio , Compostos de Magnésio/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Fosfatos/química , Fosfatos/metabolismo , Fosfoglucomutase/metabolismo , Fosforanos/metabolismo , Ligação Proteica , Conformação ProteicaRESUMO
In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP(C)). Numerous ligands have been reported to bind to human PrP(C) (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 11 complex via the structured C terminus of huPrP with a K(d) of 4.5 ± 2 µM, which is in the range of its IC(50) for curing prion-infected cells of 1.6 ± 0.4 µM and the concentration required to inhibit protein-misfolding cyclic amplification. Therefore, this molecule tests the hypothesis that stabilization of huPrP(C), as a principle, could be used in the treatment of human prion disease. The identification of a binding site with a defined 3D structure opens up the possibility of designing small molecules that stabilize huPrP and prevent its conversion into the disease-associated form.
Assuntos
Descoberta de Drogas/métodos , Modelos Moleculares , Doenças Priônicas/tratamento farmacológico , Príons/metabolismo , Ligação Proteica , Tetrapirróis/metabolismo , Sítios de Ligação/genética , Biofísica/métodos , Dicroísmo Circular , Humanos , Espectroscopia de Ressonância Magnética , Príons/química , Dobramento de Proteína , UltracentrifugaçãoRESUMO
BIRD's eye view: Adding periodic BIRD J-refocusing (BIRD=bilinear rotation decoupling) to data acquisition in an HSQC experiment causes broadband homonuclear decoupling, giving a single signal for each proton chemical shift. This pure shift method improves both resolution and signal-to-noise ratio, without the need for special data processing.