RESUMO
Structural characterization of protein-ligand binding interfaces at atomic resolution is essential for improving the design of specific and potent inhibitors. Herein, we explored fast 19F- and 1H-detected magic angle spinning NMR spectroscopy to investigate the interaction between two fluorinated ligand diastereomers with the microcrystalline galectin-3 carbohydrate recognition domain. The detailed environment around the fluorine atoms was mapped by 2D 13C-19F and 1H-19F dipolar correlation experiments and permitted characterization of the binding interface. Our results demonstrate that 19F MAS NMR is a powerful tool for detailed characterization of protein-ligand interfaces and protein interactions at the atomic level.
Assuntos
Flúor , Galectina 3 , Galectinas , Galectina 3/química , Galectina 3/metabolismo , Ligantes , Flúor/química , Galectinas/química , Galectinas/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Humanos , Ligação Proteica , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Modelos Moleculares , Domínios Proteicos , Carboidratos/químicaRESUMO
Reverse transcription, an essential event in the HIV-1 life cycle, requires deoxynucleotide triphosphates (dNTPs) to fuel DNA synthesis, thus requiring penetration of dNTPs into the viral capsid. The central cavity of the capsid protein (CA) hexamer reveals itself as a plausible channel that allows the passage of dNTPs into assembled capsids. Nevertheless, the molecular mechanism of nucleotide import into the capsid remains unknown. Employing all-atom molecular dynamics (MD) simulations, we established that cooperative binding between nucleotides inside a CA hexamer cavity results in energetically favorable conditions for passive translocation of dNTPs into the HIV-1 capsid. Furthermore, binding of the host cell metabolite inositol hexakisphosphate (IP6) enhances dNTP import, while binding of synthesized molecules like benzenehexacarboxylic acid (BHC) inhibits it. The enhancing effect on reverse transcription by IP6 and the consequences of interactions between CA and nucleotides were corroborated using atomic force microscopy, transmission electron microscopy, and virological assays. Collectively, our results provide an atomistic description of the permeability of the HIV-1 capsid to small molecules and reveal a novel mechanism for the involvement of metabolites in HIV-1 capsid stabilization, nucleotide import, and reverse transcription.
Assuntos
Capsídeo/metabolismo , HIV-1/metabolismo , Replicação Viral/fisiologia , Capsídeo/química , Capsídeo/fisiologia , Proteínas do Capsídeo/genética , Replicação do DNA/fisiologia , DNA Viral/metabolismo , Células HEK293 , HIV-1/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Simulação de Dinâmica Molecular , Nucleotídeos/metabolismo , Permeabilidade , Ácido Fítico/análise , Ácido Fítico/metabolismo , Vírion/genética , Montagem de Vírus/fisiologia , Replicação Viral/genéticaRESUMO
The nucleocapsid (N) protein is one of the four structural proteins of the SARS-CoV-2 virus and plays a crucial role in viral genome organization and, hence, replication and pathogenicity. The N-terminal domain (NNTD) binds to the genomic RNA and thus comprises a potential target for inhibitor and vaccine development. We determined the atomic-resolution structure of crystalline NNTD by integrating solid-state magic angle spinning (MAS) NMR and X-ray diffraction. Our combined approach provides atomic details of protein packing interfaces as well as information about flexible regions as the N- and C-termini and the functionally important RNA binding, ß-hairpin loop. In addition, ultrafast (100 kHz) MAS 1H-detected experiments permitted the assignment of side-chain proton chemical shifts not available by other means. The present structure offers guidance for designing therapeutic interventions against the SARS-CoV-2 infection.
Assuntos
COVID-19 , SARS-CoV-2 , Genoma Viral , Humanos , Proteínas do Nucleocapsídeo/química , RNARESUMO
19F magic angle spinning (MAS) NMR spectroscopy is a powerful tool for characterization of fluorinated solids. The recent development of 19F MAS NMR probes, operating at spinning frequencies of 60-111 kHz, enabled analysis of systems spanning from organic molecules to pharmaceutical formulations to biological assemblies, with unprecedented resolution. Herein, we systematically evaluate the benefits of high MAS frequencies (60-111 kHz) for 1D and 2D 19F-detected experiments in two pharmaceuticals, the antimalarial drug mefloquine and a formulation of the cholesterol-lowering drug atorvastatin calcium. We demonstrate that 1H decoupling is essential and that scalar-based, heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum coherence (HMQC) correlation experiments become feasible and efficient at the MAS frequency of 100 kHz. This study opens doors for the applications of high frequency 19F MAS NMR to a wide range of problems in chemistry and biology.
Assuntos
Imageamento por Ressonância Magnética , Composição de Medicamentos , Espectroscopia de Ressonância Magnética/métodosRESUMO
Fluorinated drugs occupy a large and growing share of the pharmaceutical market. Here, we explore high-frequency, 60 to 111 kHz, 19F magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy for the structural characterization of fluorinated active pharmaceutical ingredients in commercial formulations of seven blockbuster drugs: Celebrex, Cipro, Crestor, Levaquin, Lipitor, Prozac, and Zyvox. 19F signals can be observed in a single scan, and spectra with high signal-to-noise ratios can be acquired in minutes. 19F spectral parameters, such as chemical shifts and line widths, are sensitive to both the nature of the fluorine moiety and the formulation. We anticipate that the fast 19F MAS NMR-based approach presented here will be valuable for the rapid analysis of fluorine-containing drugs in a wide variety of formulations.
Assuntos
Imageamento por Ressonância Magnética , Preparações Farmacêuticas , Atorvastatina , Flúor , Espectroscopia de Ressonância MagnéticaRESUMO
Fluorine-containing compounds comprise 20 to 30 percent of all commercial drugs, and the proportion of fluorinated pharmaceuticals is rapidly growing. While magic angle spinning (MAS) NMR spectroscopy is a popular technique for analysis of solid pharmaceutical compounds, fluorine has been underutilized as a structural probe so far. Here, we report a fast (40-60 kHz) MAS 19F NMR approach for structural characterization of fluorine-containing crystalline pharmaceutical compounds at natural abundance, using the antimalarial fluorine-containing drug mefloquine as an example. We demonstrate the utility of 2D 19F-13C and 19F-19F dipolar-coupling-based correlation experiments for 19F and 13C resonance frequency assignment, which permit identification of crystallographically inequivalent sites. The efficiency of 19F-13C cross-polarization and the effect of 1H and 19F decoupling on spectral resolution and sensitivity were evaluated in a broad range of experimental conditions. We further demonstrate a protocol for measuring accurate interfluorine distances based on 1D DANTE-RFDR experiments combined with multispin numerical simulations.
Assuntos
Flúor , Preparações Farmacêuticas , Cristalografia , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância MagnéticaRESUMO
The host factor protein TRIM5α plays an important role in restricting the host range of HIV-1, interfering with the integrity of the HIV-1 capsid. TRIM5 triggers an antiviral innate immune response by functioning as a capsid pattern recognition receptor, although the precise mechanism by which the restriction is imposed is not completely understood. Here we used an integrated magic-angle spinning nuclear magnetic resonance and molecular dynamics simulations approach to characterize, at atomic resolution, the dynamics of the capsid's hexameric and pentameric building blocks, and the interactions with TRIM5α in the assembled capsid. Our data indicate that assemblies in the presence of the pentameric subunits are more rigid on the microsecond to millisecond timescales than tubes containing only hexamers. This feature may be of key importance for controlling the capsid's morphology and stability. In addition, we found that TRIM5α binding to capsid induces global rigidification and perturbs key intermolecular interfaces essential for higher-order capsid assembly, with structural and dynamic changes occurring throughout the entire CA polypeptide chain in the assembly, rather than being limited to a specific protein-protein interface. Taken together, our results suggest that TRIM5α uses several mechanisms to destabilize the capsid lattice, ultimately inducing its disassembly. Our findings add to a growing body of work indicating that dynamic allostery plays a pivotal role in capsid assembly and HIV-1 infectivity.
Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , HIV-1/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , HIV-1/genética , HIV-1/ultraestrutura , Humanos , Macaca mulatta , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína LigasesRESUMO
Chemical shift tensors obtained from solid-state NMR spectroscopy are very sensitive reporters of structure and dynamics in proteins. While accurate 13 C and 15 N chemical shift tensors are accessible by magic angle spinning (MAS) NMR, their quantum mechanical calculations remain challenging, particularly for 15 N atoms. Here we compare experimentally determined backbone 13 Cα and 15 NH chemical shift tensors by MAS NMR with hybrid quantum mechanics/molecular mechanics/molecular dynamics (MD-QM/MM) calculations for the carbohydrate-binding domain of galectin-3. Excellent agreement between experimental and computed 15 NH chemical shift anisotropy values was obtained using the Amber ff15ipq force field when solvent dynamics was taken into account in the calculation. Our results establish important benchmark conditions for improving the accuracy of chemical shift calculations in proteins and may aid in the validation of protein structure models derived by MAS NMR.
Assuntos
Proteínas Sanguíneas/química , Galectinas/química , Isótopos de Carbono/química , Teoria da Densidade Funcional , Humanos , Modelos Químicos , Simulação de Dinâmica Molecular , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear BiomolecularRESUMO
Magic angle spinning (MAS) NMR is a powerful method for the study of pharmaceutical compounds, and probes with spinning frequencies above 100 kHz enable an atomic-resolution analysis of sub-micromole quantities of fully protonated solids. Here, we present an ultrafast NMR crystallography approach for structural characterization of organic solids at MAS frequencies of 100-111 kHz. We assess the efficiency of 1H-detected experiments in the solid state and demonstrate the utility of 2D and 3D homo- and heteronuclear correlation spectra for resonance assignments. These experiments are demonstrated for an amino acid, U-13C,15N-histidine, and also for the significantly larger, natural product Posaconazole, an antifungal compound investigated at natural abundance. Our results illustrate the power for characterizing organic molecules, enabled by exploiting the increased 1H resolution and sensitivity at MAS frequencies above 100 kHz.
Assuntos
Antifúngicos/química , Histidina/química , Espectroscopia de Prótons por Ressonância Magnética/métodos , Triazóis/química , Isótopos de Carbono , Cristalografia por Raios X/métodos , Hidrogênio/química , Ligação de Hidrogênio , Imageamento por Ressonância Magnética/métodos , Isótopos de Nitrogênio , PrótonsRESUMO
Microtubule (MT)-associated proteins perform diverse functions in cells. These functions are dependent on their interactions with MTs. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150Glued, contains an N-terminus segment that is responsible for the MT-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150Glued N-terminal region, both free and in complex with polymerized MTs. This 191-residue region encompasses the cytoskeleton-associated protein glycine-rich domain, the basic domain, and serine/proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to MTs as these regions contain the second MT-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random coil, whereas the segments S111-I116, A124-R132, and K144-T146 in the basic domain contain short α-helical or ß-sheet structures. These three segments possibly encompass the MT-binding site. Surprisingly, the protein retains a high degree of flexibility upon binding to MTs except for the regions that are directly involved in the binding interactions with MTs. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.
Assuntos
Complexo Dinactina/química , Microtúbulos/química , Microtúbulos/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Complexo Dinactina/metabolismo , Espectroscopia de Ressonância Magnética , Microtúbulos/ultraestrutura , Polimerização , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Soluções , Temperatura , Tubulina (Proteína)/químicaRESUMO
In recent years, exciting developments in instrument technology and experimental methodology have advanced the field of magic-angle spinning (MAS) nuclear magnetic resonance (NMR) to new heights. Contemporary MAS NMR yields atomic-level insights into structure and dynamics of an astounding range of biological systems, many of which cannot be studied by other methods. With the advent of fast MAS, proton detection, and novel pulse sequences, large supramolecular assemblies, such as cytoskeletal proteins and intact viruses, are now accessible for detailed analysis. In this review, we will discuss the current MAS NMR methodologies that enable characterization of complex biomolecular systems and will present examples of applications to several classes of assemblies comprising bacterial and mammalian cytoskeleton as well as human immunodeficiency virus 1 and bacteriophage viruses. The body of work reviewed herein is representative of the recent advancements in the field, with respect to the complexity of the systems studied, the quality of the data, and the significance to the biology.
Assuntos
Ressonância Magnética Nuclear Biomolecular/métodos , Animais , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Humanos , Vírion/química , Vírion/metabolismoRESUMO
Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900â¯MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.
Assuntos
Proteínas do Capsídeo/química , Espectroscopia de Ressonância Magnética/métodos , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Proteínas do Capsídeo/análise , Radicais Livres/química , Espectroscopia de Ressonância Magnética/instrumentação , Proteínas de Membrana/análise , Micro-Ondas , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/instrumentação , Peptídeos/análise , TemperaturaRESUMO
We report remarkably high, up to 100-fold, signal enhancements in 19F dynamic nuclear polarization (DNP) magic angle spinning (MAS) spectra at 14.1 T on HIV-1 capsid protein (CA) assemblies. These enhancements correspond to absolute sensitivity ratios of 12-29 and are of similar magnitude to those seen for 1H signals in the same samples. At MAS frequencies above 20 kHz, it was possible to record 2D 19F-13C HETCOR spectra, which contain long-range intra- and intermolecular correlations. Such correlations provide unique distance restraints, inaccessible in conventional experiments without DNP, for protein structure determination. Furthermore, systematic quantification of the DNP enhancements as a function of biradical concentration, MAS frequency, temperature, and microwave power is reported. Our work establishes the power of DNP-enhanced 19F MAS NMR spectroscopy for structural characterization of HIV-1 CA assemblies, and this approach is anticipated to be applicable to a wide range of large biomolecular systems.
Assuntos
Proteínas do Capsídeo/química , HIV-1 , Ressonância Magnética Nuclear Biomolecular , Modelos Moleculares , Conformação ProteicaRESUMO
We present 19F longitudinal and transverse relaxation studies for four differently fluorosubstituted L-tryptophans, which carry single F atoms in the indole ring, both in the context of the free amino acid and when located in the cyclophilin A protein. For the free 4F-, 5F-, 6F-, 7F-L-Trp, satisfactory agreement between experimentally measured and calculated relaxation rates was obtained, suggesting that the parameters used for calculating the rates for the indole frame are sufficiently accurate. We also measured and calculated relaxation rates for four differently 19F-tryptophan labeled cyclophilin A proteins, transferring the parameters from the free amino acid to the protein-bound moiety. Our results suggest that 19F relaxation data of the large and rigid indole ring in Trp are only moderately affected by protein motions and provide critical reference points for evaluating fluorine NMR relaxation in the future, especially in fluorotryptophan labeled proteins.
Assuntos
Flúor/química , Ressonância Magnética Nuclear Biomolecular/métodos , Triptofano/química , Ciclofilinas/química , Indóis , Conformação ProteicaRESUMO
We present a systematic investigation into the attainable accuracy and precision of protein structures determined by heteronuclear magic angle spinning solid-state NMR for a set of four proteins of varied size and secondary structure content. Structures were calculated using synthetically generated random sets of C-C distances up to 7 Å at different degrees of completeness. For single-domain proteins, 9-15 restraints per residue are sufficient to derive an accurate model structure, while maximum accuracy and precision are reached with over 15 restraints per residue. For multi-domain proteins and protein assemblies, additional information on domain orientations, quaternary structure and/or protein shape is needed. As demonstrated for the HIV-1 capsid protein assembly, this can be accomplished by integrating MAS NMR with cryoEM data. In all cases, inclusion of TALOS-derived backbone torsion angles improves the accuracy for small number of restraints, while no further increases are noted for restraint completeness above 40%. In contrast, inclusion of TALOS-derived torsion angle restraints consistently increases the precision of the structural ensemble at all degrees of distance restraint completeness.
Assuntos
Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas/química , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Ressonância Magnética Nuclear Biomolecular/métodos , Reprodutibilidade dos TestesRESUMO
Chemical shifts are highly sensitive probes of local conformation and overall structure. Both isotropic shifts and chemical shift tensors are readily accessible from NMR experiments but their quantum mechanical calculations remain challenging. In this work, we report and compare accurately measured and calculated 15NH and 13Cα chemical shift tensors in proteins, using the microcrystalline agglutinin from Oscillatoria agardhii (OAA). Experimental 13Cα and 15NH chemical tensors were obtained by solid-state NMR spectroscopy, employing tailored recoupling sequences, and for their quantum mechanics/molecular mechanics (QM/MM) calculations different sets of functionals were evaluated. We show that 13Cα chemical shift tensors are primarily determined by backbone dihedral angles and dynamics, while 15NH tensors mainly depend on local electrostatic contributions from solvation and hydrogen bonding. In addition, the influence of including crystallographic waters, the molecular mechanics geometry optimization protocol, and the level of theory on the accuracy of the calculated chemical shift tensors is discussed. Specifically, the power of QM/MM calculations in accurately predicting the unusually upfield shifted 1HN G26 and G93 resonances is highlighted. Our integrated approach is expected to benefit structure refinement of proteins and protein assemblies.
Assuntos
Aglutininas/química , Proteínas de Bactérias/química , Espectroscopia de Ressonância Magnética/métodos , Simulação de Dinâmica Molecular , Fenômenos Biomecânicos , Cristalização , Cianobactérias/química , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Teoria Quântica , Eletricidade EstáticaRESUMO
Chemical shift tensors (CSTs) are an exquisite probe of local geometric and electronic structure. 15N CST are very sensitive to hydrogen bonding, yet they have been reported for very few proteins to date. Here we present experimental results and statistical analysis of backbone amide 15N CSTs for 100 residues of four proteins, two E. coli thioredoxin reassemblies (1-73-(U-13C,15N)/74-108-(U-15N) and 1-73-(U-15N)/74-108-(U-13C,15N)), dynein light chain 8 LC8, and CAP-Gly domain of the mammalian dynactin. The 15N CSTs were measured by a symmetry-based CSA recoupling method, ROCSA. Our results show that the principal component δ11 is very sensitive to the presence of hydrogen bonding interactions due to its unique orientation in the molecular frame. The downfield chemical shift change of backbone amide nitrogen nuclei with increasing hydrogen bond strength is manifested in the negative correlation of the principal components with hydrogen bond distance for both α-helical and ß-sheet secondary structure elements. Our findings highlight the potential for the use of 15N CSTs in protein structure refinement.
Assuntos
Amidas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/química , Animais , Ligação de HidrogênioRESUMO
Electronic and structural properties of short-lived metal-peroxido complexes, which are key intermediates in many enzymatic reactions, are not fully understood. While detected in various enzymes, their catalytic properties remain elusive because of their transient nature, making them difficult to study spectroscopically. We integrated 17O solid-state NMR and density functional theory (DFT) to directly detect and characterize the peroxido ligand in a bioinorganic V(V) complex mimicking intermediates non-heme vanadium haloperoxidases. 17O chemical shift and quadrupolar tensors, measured by solid-state NMR spectroscopy, probe the electronic structure of the peroxido ligand and its interaction with the metal. DFT analysis reveals the unusually large chemical shift anisotropy arising from the metal orbitals contributing towards the magnetic shielding of the ligand. The results illustrate the power of an integrated approach for studies of oxygen centers in enzyme reaction intermediates.
Assuntos
Complexos de Coordenação/química , Teoria da Densidade Funcional , Vanádio/química , Materiais Biomiméticos/química , Espectroscopia de Ressonância Magnética , Peroxidases/metabolismoRESUMO
Microtubules and their associated proteins perform a broad array of essential physiological functions, including mitosis, polarization and differentiation, cell migration, and vesicle and organelle transport. As such, they have been extensively studied at multiple levels of resolution (e.g., from structural biology to cell biology). Despite these efforts, there remain significant gaps in our knowledge concerning how microtubule-binding proteins bind to microtubules, how dynamics connect different conformational states, and how these interactions and dynamics affect cellular processes. Structures of microtubule-associated proteins assembled on polymeric microtubules are not known at atomic resolution. Here, we report a structure of the cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin motor on polymeric microtubules, solved by magic angle spinning NMR spectroscopy. We present the intermolecular interface of CAP-Gly with microtubules, derived by recording direct dipolar contacts between CAP-Gly and tubulin using double rotational echo double resonance (dREDOR)-filtered experiments. Our results indicate that the structure adopted by CAP-Gly varies, particularly around its loop regions, permitting its interaction with multiple binding partners and with the microtubules. To our knowledge, this study reports the first atomic-resolution structure of a microtubule-associated protein on polymeric microtubules. Our approach lays the foundation for atomic-resolution structural analysis of other microtubule-associated motors.