RESUMEN
Arrestins have pivotal roles in regulating G protein-coupled receptor (GPCR) signalling by desensitizing G protein activation and mediating receptor internalization1,2. It has been proposed that the arrestin binds to the receptor in two different conformations, 'tail' and 'core', which were suggested to govern distinct processes of receptor signalling and trafficking3,4. However, little structural information is available for the tail engagement of the arrestins. Here we report two structures of the glucagon receptor (GCGR) bound to ß-arrestin 1 (ßarr1) in glucagon-bound and ligand-free states. These structures reveal a receptor tail-engaged binding mode of ßarr1 with many unique features, to our knowledge, not previously observed. Helix VIII, instead of the receptor core, has a major role in accommodating ßarr1 by forming extensive interactions with the central crest of ßarr1. The tail-binding pose is further defined by a close proximity between the ßarr1 C-edge and the receptor helical bundle, and stabilized by a phosphoinositide derivative that bridges ßarr1 with helices I and VIII of GCGR. Lacking any contact with the arrestin, the receptor core is in an inactive state and loosely binds to glucagon. Further functional studies suggest that the tail conformation of GCGR-ßarr governs ßarr recruitment at the plasma membrane and endocytosis of GCGR, and provides a molecular basis for the receptor forming a super-complex simultaneously with G protein and ßarr to promote sustained signalling within endosomes. These findings extend our knowledge about the arrestin-mediated modulation of GPCR functionalities.
Asunto(s)
Receptores de Glucagón , beta-Arrestina 1 , beta-Arrestina 1/química , beta-Arrestina 1/metabolismo , Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Glucagón/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Ligandos , Fosfatidilinositoles/metabolismo , Receptores de Glucagón/química , Receptores de Glucagón/metabolismo , Unión ProteicaRESUMEN
Neuromedin U receptor 2 (NMU2), an emerging attractive target for treating obesity, has shown the capability in reducing food intake and regulating energy metabolism when activated. However, drug development of NMU2 was deferred partially due to the lack of structural information. Here, we present the cryo-electron microscopy (cryo-EM) structure of NMU2 bound to the endogenous agonist NmU-25 and Gi1 at 3.3 Å resolution. Combined with functional and computational data, the structure reveals the key factors that govern the recognition and selectivity of peptide agonist as well as non-peptide antagonist, providing the structural basis for design of novel and highly selective drugs targeting NMU2. In addition, a 25-degree rotation of Gi protein in reference to NMU2 is also observed compared in other structures of class A GPCR-Gi complexes, suggesting heterogeneity in the processes of G protein-coupled receptors (GPCRs) activation and G protein coupling.
Asunto(s)
Receptores Acoplados a Proteínas G , Receptores de Neurotransmisores , Ligandos , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neurotransmisores/metabolismo , Proteínas de Unión al GTP/metabolismoRESUMEN
As the only member of the CX3C chemokine receptor subfamily, CX3CR1 binds to its sole endogenous ligand CX3CL1, which shows notable potential as a therapeutic target in atherosclerosis, cancer, and neuropathy. However, the drug development of CX3CR1 is hampered partially by the lack of structural information. Here, we present two cryo-electron microscopy structures of CX3CR1-Gi1 complexes in ligand-free and CX3CL1-bound states at 2.8- and 3.4-Å resolution, respectively. Together with functional data, the structures reveal the key factors that govern the recognition of CX3CL1 by both CX3CR1 and US28. A much smaller conformational change of helix VI upon activation than previously solved class A GPCR-Gi complex structures is observed in CX3CR1, which may correlate with three cholesterol molecules that play essential roles in conformation stabilization and signaling transduction. Thus, our data deepen the understanding of cholesterol modulation in GPCR (G protein-coupled receptor) signaling and provide insights into the diversity of G protein coupling.
Asunto(s)
Quimiocina CX3CL1 , Receptores de Quimiocina , Receptor 1 de Quimiocinas CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Colesterol , Microscopía por Crioelectrón , Humanos , Receptores de Quimiocina/metabolismo , Transducción de SeñalRESUMEN
Somatostatin receptors (SSTRs) play versatile roles in inhibiting the secretion of multiple hormones such as growth hormone and thyroid-stimulating hormone, and thus are considered as targets for treating multiple tumors. Despite great progress made in therapeutic development against this diverse receptor family, drugs that target SSTRs still show limited efficacy with preferential binding affinity and conspicuous side-effects. Here, we report five structures of SSTR2 and SSTR4 in different states, including two crystal structures of SSTR2 in complex with a selective peptide antagonist and a non-peptide agonist, respectively, a cryo-electron microscopy (cryo-EM) structure of Gi1-bound SSTR2 in the presence of the endogenous ligand SST-14, as well as two cryo-EM structures of Gi1-bound SSTR4 in complex with SST-14 and a small-molecule agonist J-2156, respectively. By comparison of the SSTR structures in different states, molecular mechanisms of agonism and antagonism were illustrated. Together with computational and functional analyses, the key determinants responsible for ligand recognition and selectivity of different SSTR subtypes and multiform binding modes of peptide and non-peptide ligands were identified. Insights gained in this study will help uncover ligand selectivity of various SSTRs and accelerate the development of new molecules with better efficacy by targeting SSTRs.
Asunto(s)
Neoplasias , Receptores de Somatostatina , Microscopía por Crioelectrón , Humanos , Ligandos , Neoplasias/metabolismo , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Somatostatina/farmacología , Somatostatina/uso terapéuticoRESUMEN
In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein-coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y1, Y2, and Y4 receptors in complex with NPY or PP, and the Gi1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y1 receptor, but not with the Y2 and Y4 receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.
RESUMEN
Formyl peptide receptor 2 (FPR2) has been shown to mediate the cytotoxic effects of the ß amyloid peptide Aß42 and serves as a receptor for humanin, a peptide that protects neuronal cells from damage by Aß42, implying its involvement in the pathogenesis of Alzheimer's disease (AD). However, the interaction pattern between FPR2 and Aß42 or humanin remains unknown. Here we report the structures of FPR2 bound to Gi and Aß42 or N-formyl humanin (fHN). Combined with functional data, the structures reveal two critical regions that govern recognition and activity of Aß42 and fHN, including a polar binding cavity within the receptor helical bundle and a hydrophobic binding groove in the extracellular region. In addition, the structures of FPR2 and FPR1 in complex with different formyl peptides were determined, providing insights into ligand recognition and selectivity of the FPR family. These findings uncover key factors that define the functionality of FPR2 in AD and other inflammatory diseases and would enable drug development.
Asunto(s)
Neuroprotección , Receptores de Formil Péptido/química , Receptores de Lipoxina/química , Péptidos beta-Amiloides , Péptidos y Proteínas de Señalización Intracelular , Receptores de Formil Péptido/metabolismoRESUMEN
Adhesion G protein-coupled receptors (aGPCRs) are essential for a variety of physiological processes such as immune responses, organ development, cellular communication, proliferation and homeostasis1-7. An intrinsic manner of activation that involves a tethered agonist in the N-terminal region of the receptor has been proposed for the aGPCRs8,9, but its molecular mechanism remains elusive. Here we report the G protein-bound structures of ADGRD1 and ADGRF1, which exhibit many unique features with regard to the tethered agonism. The stalk region that proceeds the first transmembrane helix acts as the tethered agonist by forming extensive interactions with the transmembrane domain; these interactions are mostly conserved in ADGRD1 and ADGRF1, suggesting that a common stalk-transmembrane domain interaction pattern is shared by members of the aGPCR family. A similar stalk binding mode is observed in the structure of autoproteolysis-deficient ADGRF1, supporting a cleavage-independent manner of receptor activation. The stalk-induced activation is facilitated by a cascade of inter-helix interaction cores that are conserved in positions but show sequence variability in these two aGPCRs. Furthermore, the intracellular region of ADGRF1 contains a specific lipid-binding site, which proves to be functionally important and may serve as the recognition site for the previously discovered endogenous ADGRF1 ligand synaptamide. These findings highlight the diversity and complexity of the signal transduction mechanisms of the aGPCRs.
Asunto(s)
Receptores Acoplados a Proteínas G , Transducción de Señal , Humanos , Ligandos , Proteínas Oncogénicas/agonistas , Proteínas Oncogénicas/metabolismo , Unión Proteica , Dominios Proteicos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of Gi1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation.
Asunto(s)
Quimiocinas/química , Quimiocinas/metabolismo , Receptores CCR5/química , Receptores CCR5/metabolismo , Sitios de Unión , Quimiocina CCL3/metabolismo , Quimiocina CCL5/química , Quimiocina CCL5/metabolismo , Microscopía por Crioelectrón , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores CCR5/genéticaRESUMEN
The metabotropic glutamate receptors (mGlus) are involved in the modulation of synaptic transmission and neuronal excitability in the central nervous system1. These receptors probably exist as both homo- and heterodimers that have unique pharmacological and functional properties2-4. Here we report four cryo-electron microscopy structures of the human mGlu subtypes mGlu2 and mGlu7, including inactive mGlu2 and mGlu7 homodimers; mGlu2 homodimer bound to an agonist and a positive allosteric modulator; and inactive mGlu2-mGlu7 heterodimer. We observed a subtype-dependent dimerization mode for these mGlus, as a unique dimer interface that is mediated by helix IV (and that is important for limiting receptor activity) exists only in the inactive mGlu2 structure. The structures provide molecular details of the inter- and intra-subunit conformational changes that are required for receptor activation, which distinguish class C G-protein-coupled receptors from those in classes A and B. Furthermore, our structure and functional studies of the mGlu2-mGlu7 heterodimer suggest that the mGlu7 subunit has a dominant role in controlling dimeric association and G-protein activation in the heterodimer. These insights into mGlu homo- and heterodimers highlight the complex landscape of mGlu dimerization and activation.
Asunto(s)
Receptores de Glutamato Metabotrópico/química , Microscopía por Crioelectrón , Humanos , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
The metabotropic glutamate receptors (mGlus) have key roles in modulating cell excitability and synaptic transmission in response to glutamate (the main excitatory neurotransmitter in the central nervous system)1. It has previously been suggested that only one receptor subunit within an mGlu homodimer is responsible for coupling to G protein during receptor activation2. However, the molecular mechanism that underlies the asymmetric signalling of mGlus remains unknown. Here we report two cryo-electron microscopy structures of human mGlu2 and mGlu4 bound to heterotrimeric Gi protein. The structures reveal a G-protein-binding site formed by three intracellular loops and helices III and IV that is distinct from the corresponding binding site in all of the other G-protein-coupled receptor (GPCR) structures. Furthermore, we observed an asymmetric dimer interface of the transmembrane domain of the receptor in the two mGlu-Gi structures. We confirmed that the asymmetric dimerization is crucial for receptor activation, which was supported by functional data; this dimerization may provide a molecular basis for the asymmetric signal transduction of mGlus. These findings offer insights into receptor signalling of class C GPCRs.
Asunto(s)
Proteínas de Unión al GTP/química , Receptores de Glutamato Metabotrópico/química , Sitios de Unión , Microscopía por Crioelectrón , Humanos , Multimerización de Proteína , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design.
Asunto(s)
Receptores de Neuropéptido Y/metabolismo , Benzamidas/farmacología , Cristalografía por Rayos X , Células HEK293 , Humanos , Mutagénesis/genética , Mutagénesis/fisiología , Hormonas Peptídicas/farmacología , Piperazinas/farmacología , Estructura Secundaria de Proteína , Piridinas/farmacología , Receptores de Neuropéptido Y/genética , Difracción de Rayos XRESUMEN
The human formyl peptide receptor 2 (FPR2) plays a crucial role in host defense and inflammation, and has been considered as a drug target for chronic inflammatory diseases. A variety of peptides with different structures and origins have been characterized as FPR2 ligands. However, the ligand-binding modes of FPR2 remain elusive, thereby limiting the development of potential drugs. Here we report the crystal structure of FPR2 bound to the potent peptide agonist WKYMVm at 2.8 Å resolution. The structure adopts an active conformation and exhibits a deep ligand-binding pocket. Combined with mutagenesis, ligand binding and signaling studies, key interactions between the agonist and FPR2 that govern ligand recognition and receptor activation are identified. Furthermore, molecular docking and functional assays reveal key factors that may define binding affinity and agonist potency of formyl peptides. These findings deepen our understanding about ligand recognition and selectivity mechanisms of the formyl peptide receptor family.
Asunto(s)
Receptores de Formil Péptido/química , Receptores de Formil Péptido/metabolismo , Receptores de Lipoxina/química , Receptores de Lipoxina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Mutación/genética , N-Formilmetionina Leucil-Fenilalanina/química , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Conformación Proteica , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Neurokinin 1 receptor (NK1R) has key regulating functions in the central and peripheral nervous systems, and NK1R antagonists such as aprepitant have been approved for treating chemotherapy-induced nausea and vomiting. However, the lack of data on NK1R structure and biochemistry has limited further drug development targeting this receptor. Here, we combine NMR spectroscopy and X-ray crystallography to provide dynamic and static characterisation of the binding mode of aprepitant in complexes with human NK1R variants. 19F-NMR showed a slow off-rate in the binding site, where aprepitant occupies multiple substates that exchange with frequencies in the millisecond range. The environment of the bound ligand is affected by the amino acid in position 2.50, which plays a key role in ligand binding and receptor signaling in class A GPCRs. Crystal structures now reveal how receptor signaling relates to the conformation of the conserved NP7.50xxY motif in transmembrane helix VII.
Asunto(s)
Aprepitant/metabolismo , Cristalografía/métodos , Espectroscopía de Resonancia Magnética/métodos , Receptores de Neuroquinina-1/metabolismo , Aprepitant/química , Humanos , Unión Proteica , Receptores de Neuroquinina-1/químicaRESUMEN
Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR-SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Sitios de Unión , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Humanos , Enlace de Hidrógeno , Imidazoles/farmacología , Indoles/farmacología , Ligandos , Simulación del Acoplamiento Molecular , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/antagonistas & inhibidores , Conformación Proteica , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Tiazoles/farmacologíaRESUMEN
Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.
Asunto(s)
Arginina/análogos & derivados , Dihidropiridinas/química , Dihidropiridinas/metabolismo , Ácidos Difenilacéticos/química , Ácidos Difenilacéticos/metabolismo , Neuropéptido Y/metabolismo , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/química , Arginina/química , Arginina/metabolismo , Arginina/farmacología , Sitios de Unión , Cristalografía por Rayos X , Dihidropiridinas/farmacología , Ácidos Difenilacéticos/farmacología , Humanos , Fosfatos de Inositol/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Neuropéptido Y/química , Neuropéptido Y/farmacología , Resonancia Magnética Nuclear Biomolecular , Compuestos de Fenilurea/farmacología , Unión Proteica , Receptores de Neuropéptido Y/agonistas , Receptores de Neuropéptido Y/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Class B G-protein-coupled receptors (GPCRs), which consist of an extracellular domain (ECD) and a transmembrane domain (TMD), respond to secretin peptides to play a key part in hormonal homeostasis, and are important therapeutic targets for a variety of diseases. Previous work has suggested that peptide ligands bind to class B GPCRs according to a two-domain binding model, in which the C-terminal region of the peptide targets the ECD and the N-terminal region of the peptide binds to the TMD binding pocket. Recently, three structures of class B GPCRs in complex with peptide ligands have been solved. These structures provide essential insights into peptide ligand recognition by class B GPCRs. However, owing to resolution limitations, the specific molecular interactions for peptide binding to class B GPCRs remain ambiguous. Moreover, these previously solved structures have different ECD conformations relative to the TMD, which introduces questions regarding inter-domain conformational flexibility and the changes required for receptor activation. Here we report the 3.0 Å-resolution crystal structure of the full-length human glucagon receptor (GCGR) in complex with a glucagon analogue and partial agonist, NNC1702. This structure provides molecular details of the interactions between GCGR and the peptide ligand. It reveals a marked change in the relative orientation between the ECD and TMD of GCGR compared to the previously solved structure of the inactive GCGR-NNC0640-mAb1 complex. Notably, the stalk region and the first extracellular loop undergo major conformational changes in secondary structure during peptide binding, forming key interactions with the peptide. We further propose a dual-binding-site trigger model for GCGR activation-which requires conformational changes of the stalk, first extracellular loop and TMD-that extends our understanding of the previously established two-domain peptide-binding model of class B GPCRs.
Asunto(s)
Glucagón/análogos & derivados , Receptores de Glucagón/química , Receptores de Glucagón/metabolismo , Cristalografía por Rayos X , Agonismo Parcial de Drogas , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
In response to adenosine 5'-diphosphate, the P2Y1 receptor (P2Y1R) facilitates platelet aggregation, and thus serves as an important antithrombotic drug target. Here we report the crystal structures of the human P2Y1R in complex with a nucleotide antagonist MRS2500 at 2.7 Å resolution, and with a non-nucleotide antagonist BPTU at 2.2 Å resolution. The structures reveal two distinct ligand-binding sites, providing atomic details of P2Y1R's unique ligand-binding modes. MRS2500 recognizes a binding site within the seven transmembrane bundle of P2Y1R, which is different in shape and location from the nucleotide binding site in the previously determined structure of P2Y12R, representative of another P2YR subfamily. BPTU binds to an allosteric pocket on the external receptor interface with the lipid bilayer, making it the first structurally characterized selective G-protein-coupled receptor (GPCR) ligand located entirely outside of the helical bundle. These high-resolution insights into P2Y1R should enable discovery of new orthosteric and allosteric antithrombotic drugs with reduced adverse effects.
Asunto(s)
Nucleótidos de Desoxiadenina/química , Nucleótidos de Desoxiadenina/metabolismo , Antagonistas del Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y1/química , Receptores Purinérgicos P2Y1/metabolismo , Uracilo/análogos & derivados , Adenosina Difosfato/análogos & derivados , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Nucleótidos de Desoxiadenina/farmacología , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Antagonistas del Receptor Purinérgico P2Y/metabolismo , Antagonistas del Receptor Purinérgico P2Y/farmacología , Tionucleótidos/química , Tionucleótidos/metabolismo , Uracilo/química , Uracilo/metabolismo , Uracilo/farmacologíaRESUMEN
Human Raf-1 kinase inhibitor protein (hRKIP) is a small multi-functional protein of 187 residues. It contains a conserved pocket, which binds a wide range of ligands from various small molecules to distinct proteins. To provide a structural basis for the ligand diversity of RKIP, we herein determined the solution structure of hRKIP, and analyzed its structural dynamics. In solution, hRKIP mainly comprises two antiparallel ß sheets, two α helices and two 310 helices. NMR dynamic analysis reveals that the overall structure of hRKIP is rigid, but its C-terminal helix which is close to the ligand-binding site is mobile. In addition, residues around the ligand-binding pocket exhibit significant conformational exchange on the µs-ms timescale. Conformational flexibility may allow the ligand-binding pocket and the C-terminal helix to adopt various conformations to interact with different substrates. This work may shed light on the underlying molecular mechanisms of how hRKIP recognizes and binds diverse substrate ligands.
Asunto(s)
Modelos Químicos , Modelos Moleculares , Proteínas de Unión a Fosfatidiletanolamina/química , Proteínas de Unión a Fosfatidiletanolamina/ultraestructura , Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conformación ProteicaRESUMEN
Raf-1 kinase inhibitor protein (RKIP) plays a pivotal role in modulating multiple signaling networks. Here we report backbone and side chain resonance assignments of uniformly (15)N, (13)C labeled human RKIP.