RESUMO
Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artifact-free dynamics measurements, and allows probing motions effectively without molecular weight limitations. The application to the TET2 enzyme assembly of â¼0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (picoseconds to milliseconds). We quantitatively probe ring-flip motions and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as the exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.
Assuntos
Aminopeptidases/química , Ressonância Magnética Nuclear Biomolecular , Termodinâmica , Aminopeptidases/metabolismo , Isótopos de Carbono , Conformação Proteica , Prótons , Pyrococcus horikoshii/enzimologiaRESUMO
Studying protein dynamics on microsecond-to-millisecond (µs-ms) time scales can provide important insight into protein function. In magic-angle-spinning (MAS) NMR, µs dynamics can be visualized by R 1 ρ rotating-frame relaxation dispersion experiments in different regimes of radio-frequency field strengths: at low RF field strength, isotropic-chemical-shift fluctuation leads to "Bloch-McConnell-type" relaxation dispersion, while when the RF field approaches rotary resonance conditions bond angle fluctuations manifest as increased R 1 ρ rate constants ("Near-Rotary-Resonance Relaxation Dispersion", NERRD). Here we explore the joint analysis of both regimes to gain comprehensive insight into motion in terms of geometric amplitudes, chemical-shift changes, populations and exchange kinetics. We use a numerical simulation procedure to illustrate these effects and the potential of extracting exchange parameters, and apply the methodology to the study of a previously described conformational exchange process in microcrystalline ubiquitin.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Proteínas/química , Conformação Proteica , Ubiquitina/químicaRESUMO
The intrinsically disordered protein domain DCL1-A is the first report of a complete double stranded RNA binding domain folding upon binding. DCL1-A recognizes the dsRNA by acquiring a well-folded structure after engagement with its interaction partner. Despite the structural characterization of the interaction complex underlying the recognition of dsRNA has been established, the dynamics of disorder-to-order transitions in the binding process remains elusive. Here we have developed a coarse-grained structure-based model with consideration of electrostatic interactions to explore the mechanism of the coupled folding and binding. Our approach led to remarkable agreements with both experimental and theoretical results. We quantified the global binding-folding landscape, which indicates a synergistic binding induced folding mechanism. We further investigated the effect of electrostatic interactions in this coupled folding and binding process. It reveals that non-native electrostatic interactions dominate the initial stage of the recognition. Our results help improve our understanding of the induced folding of the IDP DCL1-A upon binding to dsRNA. Such methods developed here can be applied for further explorations of the dynamics of coupled folding and binding systems.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Dobramento de Proteína , RNA/química , Ribonuclease III/química , Simulação por Computador , Cinética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Relação Estrutura-Atividade , TermodinâmicaRESUMO
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. Substrate pri-miRNA recognition by DCL1 requires two double stranded RNA binding domains located at the C-terminus of the protein. We have previously shown that the first of these domains, DCL1-A, is intrinsically disordered and folds upon binding pri-miRNA. Integrating NMR and SAXS data, we study here the conformational landscape of free DCL1-A through an ensemble description. Our results reveal that secondary structure elements, corresponding to the folded form of the protein, are transiently populated in the unbound state. The conformation of one of the dsRNA binding regions in the free protein shows that, at a local level, RNA recognition proceeds through a conformational selection mechanism. We further explored the stability of the preformed structural elements via temperature and urea destabilization. The C-terminal helix is halfway on the folding pathway in free DCL1-A, constituting a potential nucleation site for the final folding of the protein. In contrast, the N-terminal helix adopts stable non-native structures that could hinder the correct folding of the protein in the absence of RNA. This description of the unfolded form allows us to understand details of the mechanism of binding-induced folding of the protein.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Intrinsicamente Desordenadas/química , MicroRNAs/metabolismo , Ribonuclease III/metabolismo , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , MicroRNAs/química , Modelos Químicos , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Dobramento de Proteína , Ribonuclease III/química , Espalhamento a Baixo Ângulo , Temperatura , Difração de Raios XRESUMO
Solid-state NMR spectroscopy can provide insight into protein structure and dynamics at the atomic level without inherent protein size limitations. However, a major hurdle to studying large proteins by solid-state NMR spectroscopy is related to spectral complexity and resonance overlap, which increase with molecular weight and severely hamper the assignment process. Here the use of two sets of experiments is shown to expand the tool kit of 1 H-detected assignment approaches, which correlate a given amide pair either to the two adjacent CO-CA pairs (4D hCOCANH/hCOCAcoNH), or to the amide 1 H of the neighboring residue (3D HcocaNH/HcacoNH, which can be extended to 5D). The experiments are based on efficient coherence transfers between backbone atoms using INEPT transfers between carbons and cross-polarization for heteronuclear transfers. The utility of these experiments is exemplified with application to assemblies of deuterated, fully amide-protonated proteins from approximately 20 to 60â kDa monomer, at magic-angle spinning (MAS) frequencies from approximately 40 to 55â kHz. These experiments will also be applicable to protonated proteins at higher MAS frequencies. The resonance assignment of a domain within the 50.4â kDa bacteriophageâ T5 tube protein pb6 is reported, and this is compared to NMR assignments of the isolated domain in solution. This comparison reveals contacts of this domain to the core of the polymeric tail tube assembly.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Amidas/químicaRESUMO
Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45-60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a ß-turn region in the wild-type protein. We use 15N R1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-1H R1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of 1H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.
Assuntos
Proteínas Mutantes/química , Mutação , Ressonância Magnética Nuclear Biomolecular , Ubiquitina/química , Cristalografia por Raios X , Modelos Moleculares , Proteínas Mutantes/genética , Conformação Proteica , Ubiquitina/genéticaRESUMO
MOTIVATION: Water molecules are key players for protein folding and function. On the protein surface, water is not placed randomly, but display instead a particular structure evidenced by the presence of specific water sites (WS). These WS can be derived and characterized using explicit water Molecular Dynamics simulations, providing useful information for ligand binding prediction and design. Here we present WATCLUST, a WS determination and analysis tool running on the VMD platform. The tool also allows direct transfer of the WS information to Autodock program to perform biased docking. AVAILABILITY AND IMPLEMENTATION: The WATCLUST plugin and documentation are freely available at http://sbg.qb.fcen.uba.ar/watclust/. CONTACT: marcelo@qi.fcen.uba.ar, adrian@qi.fcen.uba.ar.
Assuntos
Desenho de Fármacos , Proteínas/química , Software , Água/química , Proteínas de Escherichia coli/química , Simulação de Dinâmica MolecularRESUMO
Double stranded RNA (dsRNA) participates in several biological processes, where RNA molecules acquire secondary structure inside the cell through base complementarity. The double stranded RNA binding domain (dsRBD) is one of the main protein folds that is able to recognize and bind to dsRNA regions. The N-terminal dsRBD of DCL1 in Arabidopsis thaliana (DCL1-1), in contrast to other studied dsRBDs, lacks a stable structure, behaving as an intrinsically disordered protein. DCL1-1 does however recognize dsRNA by acquiring a canonical fold in the presence of its substrate. Here we present a detailed modeling and molecular dynamics study of dsRNA recognition by DCL1-1. We found that DCL1-1 forms stable complexes with different RNAs and we characterized the residues involved in binding. Although the domain shows a binding loop substantially shorter than other homologs, it can still interact with the dsRNA and results in bending of the dsRNA A-type helix. Furthermore, we found that R8, a non-conserved residue located in the first dsRNA binding region, recognizes preferentially mismatched base pairs. We discuss our findings in the context of the function of DCL1-1 within the microRNA processing complex.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas de Ciclo Celular/química , MicroRNAs/química , Modelos Químicos , Simulação de Dinâmica Molecular , RNA de Cadeia Dupla/química , RNA de Plantas/química , Ribonuclease III/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , MicroRNAs/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA de Plantas/metabolismo , Ribonuclease III/metabolismoRESUMO
Recognition and complex formation between proteins and carbohydrates is a key issue in many important biological processes. Determination of the three-dimensional structure of such complexes is thus most relevant, but particularly challenging because of their usually low binding affinity. In silico docking methods have a long-standing tradition in predicting protein-ligand complexes, and allow a potentially fast exploration of a number of possible protein-carbohydrate complex structures. However, determining which of these predicted complexes represents the correct structure is not always straightforward. In this work, we present a modification of the scoring function provided by AutoDock4, a widely used docking software, on the basis of analysis of the solvent structure adjacent to the protein surface, as derived from molecular dynamics simulations, that allows the definition and characterization of regions with higher water occupancy than the bulk solvent, called water sites. They mimic the interaction held between the carbohydrate -OH groups and the protein. We used this information for an improved docking method in relation to its capacity to correctly predict the protein-carbohydrate complexes for a number of tested proteins, whose ligands range in size from mono- to tetrasaccharide. Our results show that the presented method significantly improves the docking predictions. The resulting solvent-structure-biased docking protocol, therefore, appears as a powerful tool for the design and optimization of development of glycomimetic drugs, while providing new insights into protein-carbohydrate interactions. Moreover, the achieved improvement also underscores the relevance of the solvent structure to the protein carbohydrate recognition process.
Assuntos
Carboidratos/química , Simulação de Dinâmica Molecular , Proteínas/química , Solventes/química , Sítios de Ligação , Galectinas/química , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Software , Água/química , Água/metabolismoRESUMO
Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects.
RESUMO
Large oligomeric enzymes control a myriad of cellular processes, from protein synthesis and degradation to metabolism. The 0.5 MDa large TET2 aminopeptidase, a prototypical protease important for cellular homeostasis, degrades peptides within a ca. 60 Å wide tetrahedral chamber with four lateral openings. The mechanisms of substrate trafficking and processing remain debated. Here, we integrate magic-angle spinning (MAS) NMR, mutagenesis, co-evolution analysis and molecular dynamics simulations and reveal that a loop in the catalytic chamber is a key element for enzymatic function. The loop is able to stabilize ligands in the active site and may additionally have a direct role in activating the catalytic water molecule whereby a conserved histidine plays a key role. Our data provide a strong case for the functional importance of highly dynamic - and often overlooked - parts of an enzyme, and the potential of MAS NMR to investigate their dynamics at atomic resolution.
Assuntos
Aminopeptidases , Simulação de Dinâmica Molecular , Aminopeptidases/metabolismo , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , PeptídeosRESUMO
Galectins, a family of evolutionarily conserved animal lectins, have been shown to modulate signaling processes leading to inflammation, apoptosis, immunoregulation, and angiogenesis through their ability to interact with poly-N-acetyllactosamine-enriched glycoconjugates. To date 16 human galectin carbohydrate recognition domains have been established by sequence analysis and found to be expressed in several tissues. Given the divergent functions of these lectins, it is of vital importance to understand common and differential features in order to search for specific inhibitors of individual members of the human galectin family. In this work we performed an integrated computational analysis of all individual members of the human galectin family. In the first place, we have built homology-based models for galectin-4 and -12 N-terminus, placental protein 13 (PP13) and PP13-like protein for which no experimental structural information is available. We have then performed classical molecular dynamics simulations of the whole 15 members family in free and ligand-bound states to analyze protein and protein-ligand interaction dynamics. Our results show that all galectins adopt the same fold, and the carbohydrate recognition domains are very similar with structural differences located in specific loops. These differences are reflected in the dynamics characteristics, where mobility differences translate into entropy values which significantly influence their ligand affinity. Thus, ligand selectivity appears to be modulated by subtle differences in the monosaccharide binding sites. Taken together, our results may contribute to the understanding, at a molecular level, of the structural and dynamical determinants that distinguish individual human galectins.
Assuntos
Galectina 4/análise , Galectinas/análise , Polissacarídeos/metabolismo , Proteínas da Gravidez/análise , Transdução de Sinais/fisiologia , Biologia de Sistemas/métodos , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Entropia , Epitopos , Galectina 4/química , Galectina 4/imunologia , Galectina 4/metabolismo , Galectinas/química , Galectinas/imunologia , Galectinas/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Filogenia , Polissacarídeos/imunologia , Proteínas da Gravidez/química , Proteínas da Gravidez/imunologia , Proteínas da Gravidez/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Formation of protein ligand complexes is a fundamental phenomenon in biochemistry. During the process, significant solvent reorganization is produced along the contact surface and many water molecules strongly bound to the protein's ligand binding site must be displaced. Both the thermodynamics and kinetics of this process are complex and a clear understanding at the microscopic level has been not achieved so far. Special attention has been paid to the structure of water molecules on carbohydrate recognition sites of various proteins, and many studies support the idea that displacement of these water molecules should have a crucial effect on the binding free energy. Molecular dynamics (MD) simulations in explicit water solvent is a very promising approach for this type of studies. Using MD simulations combined with statistical mechanics analysis, thermodynamic properties of these water molecules can be computed and analyzed in a comparative view. Using this idea, we developed a set of analysis tools to link solvation with ligand binding in a key carbohydrate binding protein, human galectin-1 (hGal-1). Specifically, we defined water sites (WS) in terms of the thermodynamic properties of water molecules strongly bound to protein surfaces. In the present work, we selected a group of proteins whose ligand bound complexes have been already structurally characterized in order to extend the analysis of the role of the surface associated water molecules in the ligand binding and recognition process. The selected proteins are concanavalin-A (Con-A), galectin-3 (Gal-3), cyclophilin-A (Cyp-A), and two modules CBM40 and CBM32 of the multimodular bacterial sialidase. Our results show that the probability of finding water molecules inside the WS, p(v), with respect to the bulk density is directly correlated to the likeliness of finding an hydroxyl group of the ligand in the protein-ligand complex. This information can be used to analyze in detail the solvation structure of the carbohydrate recognition domain (CRD) and its relation to the possible protein ligand complexes and suggests addition of OH-containing functional groups to displace water from high p(v) WS to enhance drugs, specially glycomimetic-drugs, protein affinity, and/or specificity.
Assuntos
Metabolismo dos Carboidratos , Modelos Moleculares , Proteínas/química , Proteínas/metabolismo , Solventes/química , Solventes/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ligantes , Conformação Proteica , Termodinâmica , Água/química , Água/metabolismoRESUMO
Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. Caseinolytic protease (ClpP) is a tetradecameric complex, which has emerged as a drug target against multiple pathogenic bacteria. Activation of different ClpPs by inhibitors has been independently reported from drug development efforts, but no rationale for inhibitor-induced activation has been hitherto proposed. Using an integrated approach that includes x-ray crystallography, solid- and solution-state nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the proteasome inhibitor bortezomib binds to the ClpP active-site serine, mimicking a peptide substrate, and induces a concerted allosteric activation of the complex. The bortezomib-activated conformation also exhibits a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism, where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association and protein processive degradation.
Assuntos
Proteínas de Bactérias , Endopeptidase Clp , Inibidores de Proteases/química , Thermus thermophilus/enzimologia , Regulação Alostérica , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Domínio Catalítico , Cristalografia por Raios X , Endopeptidase Clp/antagonistas & inibidores , Endopeptidase Clp/químicaRESUMO
Atomic-resolution structure determination is crucial for understanding protein function. Cryo-EM and NMR spectroscopy both provide structural information, but currently cryo-EM does not routinely give access to atomic-level structural data, and, generally, NMR structure determination is restricted to small (<30 kDa) proteins. We introduce an integrated structure determination approach that simultaneously uses NMR and EM data to overcome the limits of each of these methods. The approach enables structure determination of the 468 kDa large dodecameric aminopeptidase TET2 to a precision and accuracy below 1 Å by combining secondary-structure information obtained from near-complete magic-angle-spinning NMR assignments of the 39 kDa-large subunits, distance restraints from backbone amides and ILV methyl groups, and a 4.1 Å resolution EM map. The resulting structure exceeds current standards of NMR and EM structure determination in terms of molecular weight and precision. Importantly, the approach is successful even in cases where only medium-resolution cryo-EM data are available.
Assuntos
Complexos Multienzimáticos/ultraestrutura , Estrutura Quaternária de Proteína , Aminopeptidases/química , Aminopeptidases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Peso Molecular , Complexos Multienzimáticos/química , Pyrococcus horikoshiiRESUMO
Solid-state NMR spectroscopy allows the characterization of the structure, interactions and dynamics of insoluble and/or very large proteins. Sensitivity and resolution are often major challenges for obtaining atomic-resolution information, in particular for very large protein complexes. Here we show that the use of deuterated, specifically CH3-labelled proteins result in significant sensitivity gains compared to previously employed CHD2 labelling, while line widths increase only marginally. We apply this labelling strategy to a 468 kDa-large dodecameric aminopeptidase, TET2, and the 1.6 MDa-large 50S ribosome subunit of Thermus thermophilus.
RESUMO
Lectins are able to recognize specific carbohydrate structures through their carbohydrate recognition domain (CRD). The lectin from the mushroom Agaricus bisporus (ABL) has the remarkable ability of selectively recognizing the TF-antigen, composed of Galß1-3GalNAc, Ser/Thr linked to proteins, specifically exposed in neoplastic tissues. Strikingly, the recently solved crystal structure of tetrameric ABL in the presence of TF-antigen and other carbohydrates showed that each monomer has two CRDs, each being able to bind specifically to different monosaccharides that differ only in the configuration of a single hydroxyl, like N-acetyl-d-galactosamine (GalNAc) and N-acetyl-d-glucosamine (GlcNAc). Understanding how lectin CRDs bind and discriminate mono and/or (poly)-saccharides is an important issue in glycobiology, with potential impact in the design of better and selective lectin inhibitors with potential therapeutic properties. In this work, and based on the unusual monosaccharide epimeric specificity of the ABL CRDs, we have performed molecular dynamics simulations of the natural (crystallographic) and inverted (changing GalNAc for GlcNAc and vice-versa) ABL-monosaccharide complexes in order to understand the selective ligand recognition properties of each CRD. We also performed a detailed analysis of the CRD local solvent structure, using previously developed methodology, and related it with the recognition mechanism. Our results provide a detailed picture of each ABL CRD specificity, allowing a better understanding of the carbohydrate selective recognition process in this particular lectin.