RESUMEN
Biomolecules are in constant motion. To understand how they function, and why malfunctions can cause disease, it is necessary to describe their three-dimensional structures in terms of dynamic conformational ensembles. Here, we demonstrate how nuclear magnetic resonance (NMR) spectroscopy provides an essential, dynamic view of structural biology that captures biomolecular motions at atomic resolution. We focus on examples that emphasize the diversity of biomolecules and biochemical applications that are amenable to NMR, such as elucidating functional dynamics in large molecular machines, characterizing transient conformations implicated in the onset of disease, and obtaining atomic-level descriptions of intrinsically disordered regions that make weak interactions involved in liquid-liquid phase separation. Finally, we discuss the pivotal role that NMR has played in driving forward our understanding of the biomolecular dynamics-function paradigm.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Biomarcadores/metabolismo , Variaciones en el Número de Copia de ADN/genética , Humanos , Mutación/genética , Transcriptoma/genéticaRESUMEN
The RIPK1-RIPK3 necrosome is an amyloid signaling complex that initiates TNF-induced necroptosis, serving in human immune defense, cancer, and neurodegenerative diseases. RIPK1 and RIPK3 associate through their RIP homotypic interaction motifs with consensus sequences IQIG (RIPK1) and VQVG (RIPK3). Using solid-state nuclear magnetic resonance, we determined the high-resolution structure of the RIPK1-RIPK3 core. RIPK1 and RIPK3 alternately stack (RIPK1, RIPK3, RIPK1, RIPK3, etc.) to form heterotypic ß sheets. Two such ß sheets bind together along a compact hydrophobic interface featuring an unusual ladder of alternating Ser (from RIPK1) and Cys (from RIPK3). The crystal structure of a four-residue RIPK3 consensus sequence is consistent with the architecture determined by NMR. The RIPK1-RIPK3 core is the first detailed structure of a hetero-amyloid and provides a potential explanation for the specificity of hetero- over homo-amyloid formation and a structural basis for understanding the mechanisms of signal transduction.
Asunto(s)
Amiloide/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Resonancia Magnética Nuclear Biomolecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Alineación de SecuenciaRESUMEN
Dozens of proteins are known to convert to the aggregated amyloid state. These include fibrils associated with systemic and neurodegenerative diseases and cancer, functional amyloid fibrils in microorganisms and animals, and many denatured proteins. Amyloid fibrils can be much more stable than other protein assemblies. In contrast to globular proteins, a single protein sequence can aggregate into several distinctly different amyloid structures, termed polymorphs, and a given polymorph can reproduce itself by seeding. Amyloid polymorphs may be the molecular basis of prion strains. Whereas the Protein Data Bank contains some 100,000 globular protein and 3,000 membrane protein structures, only a few dozen amyloid protein structures have been determined, and most of these are short segments of full amyloid-forming proteins. Regardless, these amyloid structures illuminate the architecture of the amyloid state, including its stability and its capacity for formation of polymorphs.
Asunto(s)
Proteínas Amiloidogénicas/química , Proteínas Priónicas/química , Agregación Patológica de Proteínas/metabolismo , Secuencias de Aminoácidos , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Animales , Microscopía por Crioelectrón , Expresión Génica , Humanos , Resonancia Magnética Nuclear Biomolecular , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Desnaturalización Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Difracción de Rayos XRESUMEN
Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.
Asunto(s)
Proteína FUS de Unión a ARN/química , Secuencia de Aminoácidos , Humanos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Fosforilación , Dominios Proteicos , Proteína FUS de Unión a ARN/metabolismoRESUMEN
The essential principles of nuclear magnetic resonance for the analysis of biomolecules are conveyed in this SnapShot.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Modelos Moleculares , Ácidos Nucleicos/química , Proteínas/químicaRESUMEN
The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.
Asunto(s)
Receptores ErbB/química , Receptores ErbB/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/aislamiento & purificación , Humanos , Membranas Intracelulares/química , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Termodinámica , Vesículas Transportadoras/químicaRESUMEN
Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.
Asunto(s)
Glicina/análogos & derivados , Proteínas de Unión al ARN/química , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Cristalografía por Rayos X , Dimerización , Glicina/administración & dosificación , Glicina/química , Glicina/farmacología , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Neoplasias Pancreáticas/tratamiento farmacológico , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Sulfonas/administración & dosificación , Sulfonas/química , Proteínas ras/metabolismo , Quinasa Tipo Polo 1RESUMEN
Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on (13)C- and (15)N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and (1)H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron-nuclear transitions. DNP boosts NMR signal intensities by factors of 10(2) to 10(3), thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, (1)H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively (2)H-labeled proteins.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Amiloide/química , Bacterias/química , Humanos , Hidrógeno/análisis , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular/instrumentaciónRESUMEN
G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.
Asunto(s)
Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Benzoxazinas/farmacología , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacología , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 2/químicaRESUMEN
Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment "invisible" and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Factores de Terminación de Péptidos/química , Priones/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis1-3. Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts4,5. This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes.
Asunto(s)
Biocatálisis , Ácidos Borónicos , Enzimas , Ingeniería de Proteínas , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Cristalografía por Rayos X , Evolución Molecular Dirigida , Enzimas/química , Enzimas/metabolismo , Enzimas/genética , Cetonas/química , Cetonas/metabolismo , Cinética , Modelos Moleculares , Oximas/química , Oximas/metabolismo , Especificidad por Sustrato , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masas , Xenobióticos/química , Xenobióticos/metabolismoRESUMEN
Intermediate species in the assembly of amyloid filaments are believed to play a central role in neurodegenerative diseases and may constitute important targets for therapeutic intervention1,2. However, structural information about intermediate species has been scarce and the molecular mechanisms by which amyloids assemble remain largely unknown. Here we use time-resolved cryogenic electron microscopy to study the in vitro assembly of recombinant truncated tau (amino acid residues 297-391) into paired helical filaments of Alzheimer's disease or into filaments of chronic traumatic encephalopathy3. We report the formation of a shared first intermediate amyloid filament, with an ordered core comprising residues 302-316. Nuclear magnetic resonance indicates that the same residues adopt rigid, ß-strand-like conformations in monomeric tau. At later time points, the first intermediate amyloid disappears and we observe many different intermediate amyloid filaments, with structures that depend on the reaction conditions. At the end of both assembly reactions, most intermediate amyloids disappear and filaments with the same ordered cores as those from human brains remain. Our results provide structural insights into the processes of primary and secondary nucleation of amyloid assembly, with implications for the design of new therapies.
Asunto(s)
Enfermedad de Alzheimer , Amiloide , Encefalopatía Traumática Crónica , Ovillos Neurofibrilares , Proteínas tau , Humanos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestructura , Encefalopatía Traumática Crónica/metabolismo , Encefalopatía Traumática Crónica/patología , Microscopía por Crioelectrón , Ovillos Neurofibrilares/química , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/ultraestructura , Proteínas tau/química , Proteínas tau/metabolismo , Proteínas tau/ultraestructura , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Factores de TiempoRESUMEN
Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.
Asunto(s)
Microscopía por Crioelectrón , Péptidos y Proteínas de Señalización Intracelular , Proteínas Intrínsecamente Desordenadas , Fosfoproteínas , Proteína Fosfatasa 2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/ultraestructura , Mitosis , Resonancia Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestructura , Fosforilación , Proteína Fosfatasa 2/química , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/ultraestructuraRESUMEN
Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs to neurotransmission, little is known about the structures and dynamics of intact receptors in distinct functional states. Here, we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with partial agonists and a positive allosteric modulator, and in a desensitized/closed state in complex with fluorowilliardiine. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryoelectron microscopy studies. We show how agonist binding modulates the conformation of the ligand-binding domain "layer" of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of the amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation, and desensitization in AMPA iGluRs.
Asunto(s)
Receptores AMPA/química , Receptores AMPA/metabolismo , Animales , Microscopía por Crioelectrón , Cristalografía por Rayos X , Fluorouracilo/análogos & derivados , Fluorouracilo/metabolismo , Técnicas de Inactivación de Genes , Ácido Kaínico/metabolismo , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Ratas , Receptores AMPA/agonistas , Receptores AMPA/genéticaRESUMEN
The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates.
Asunto(s)
Chaperonina con TCP-1/química , Chaperonina con TCP-1/metabolismo , Eucariontes/química , Pliegue de Proteína , Animales , Archaea/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Bovinos , Chaperonina con TCP-1/genética , Eucariontes/citología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.
Asunto(s)
Butirofilinas , Activación de Linfocitos , Fosfoproteínas , Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos T , Animales , Humanos , Antígenos CD/inmunología , Antígenos CD/metabolismo , Butirofilinas/inmunología , Butirofilinas/metabolismo , Camélidos del Nuevo Mundo/inmunología , Simulación de Dinámica Molecular , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , TermodinámicaRESUMEN
G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the ß(2)-adrenergic receptor (ß(2)AR), a prototypical GPCR. We labeled ß(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for ß(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for ß(2)AR's ability to engage multiple signaling and regulatory proteins.
Asunto(s)
Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Transducción de Señal , TermodinámicaRESUMEN
Dimerization-driven activation of the intracellular kinase domains of the epidermal growth factor receptor (EGFR) upon extracellular ligand binding is crucial to cellular pathways regulating proliferation, migration, and differentiation. Inactive EGFR can exist as both monomers and dimers, suggesting that the mechanism regulating EGFR activity may be subtle. The membrane itself may play a role but creates substantial difficulties for structural studies. Our molecular dynamics simulations of membrane-embedded EGFR suggest that, in ligand-bound dimers, the extracellular domains assume conformations favoring dimerization of the transmembrane helices near their N termini, dimerization of the juxtamembrane segments, and formation of asymmetric (active) kinase dimers. In ligand-free dimers, by holding apart the N termini of the transmembrane helices, the extracellular domains instead favor C-terminal dimerization of the transmembrane helices, juxtamembrane segment dissociation and membrane burial, and formation of symmetric (inactive) kinase dimers. Electrostatic interactions of EGFR's intracellular module with the membrane are critical in maintaining this coupling.
Asunto(s)
Membrana Celular/metabolismo , Receptores ErbB/química , Membrana Celular/química , Dimerización , Receptores ErbB/metabolismo , Humanos , Lípidos de la Membrana/metabolismo , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Electricidad EstáticaRESUMEN
Macromolecular function frequently requires that proteins change conformation into high-energy states1-4. However, methods for solving the structures of these functionally essential, lowly populated states are lacking. Here we develop a method for high-resolution structure determination of minorly populated states by coupling NMR spectroscopy-derived pseudocontact shifts5 (PCSs) with Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion6 (PCS-CPMG). Our approach additionally defines the corresponding kinetics and thermodynamics of high-energy excursions, thereby characterizing the entire free-energy landscape. Using a large set of simulated data for adenylate kinase (Adk), calmodulin and Src kinase, we find that high-energy PCSs accurately determine high-energy structures (with a root mean squared deviation of less than 3.5 angström). Applying our methodology to Adk during catalysis, we find that the high-energy excursion involves surprisingly small openings of the AMP and ATP lids. This previously unresolved high-energy structure solves a longstanding controversy about conformational interconversions that are rate-limiting for catalysis. Primed for either substrate binding or product release, the high-energy structure of Adk suggests a two-step mechanism combining conformational selection to this state, followed by an induced-fit step into a fully closed state for catalysis of the phosphoryl-transfer reaction. Unlike other methods for resolving high-energy states, such as cryo-electron microscopy and X-ray crystallography, our solution PCS-CPMG approach excels in cases involving domain rearrangements of smaller systems (less than 60 kDa) and populations as low as 0.5%, and enables the simultaneous determination of protein structure, kinetics and thermodynamics while proteins perform their function.
Asunto(s)
Adenilato Quinasa , Adenilato Quinasa/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , TermodinámicaRESUMEN
The 10-23 DNAzyme is one of the most prominent catalytically active DNA sequences1,2. Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential2. However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action3. Here we provide high-resolution NMR characterization of all apparent states of the prototypic 10-23 DNAzyme and present a comprehensive survey of the kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites of the precatalytic DNAzyme-RNA complex reveal that the basis of the DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic process via real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance of the DNAzyme, demonstrating that the acquired knowledge of the molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.