RESUMEN
Intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs) is a class of biologically important proteins exhibiting specific biophysical characteristics. They lack a hydrophobic core, and their conformational behavior is strongly influenced by electrostatic interactions. IDPs and IDRs are highly dynamic, and a characterization of the motions of IDPs and IDRs is essential for their physically correct description. NMR together with molecular dynamics simulations are the methods best suited to such a task because they provide information about dynamics of proteins with atomistic resolution. Here, we present a study of motions of a disordered C-terminal domain of the delta subunit of RNA polymerase from Bacillus subtilis. Positively and negatively charged residues in the studied domain form transient electrostatic contacts critical for the biological function. Our study is focused on investigation of ps-ns dynamics of backbone of the delta subunit based on analysis of amide 15N NMR relaxation data and molecular dynamics simulations. In order to extend an informational content of NMR data to lower frequencies, which are more sensitive to slower motions, we combined standard (high-field) NMR relaxation experiments with high-resolution relaxometry. Altogether, we collected data reporting the relaxation at 12 different magnetic fields, resulting in an unprecedented data set. Our results document that the analysis of such data provides a consistent description of dynamics and confirms the validity of so far used protocols of the analysis of dynamics of IDPs also for a partially folded protein. In addition, the potential to access detailed description of motions at the timescale of tens of ns with the help of relaxometry data is discussed. Interestingly, in our case, it appears to be mostly relevant for a region involved in the formation of temporary contacts within the disordered region, which was previously proven to be biologically important.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas Intrínsecamente Desordenadas/química , Conformación Proteica , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , ARN Polimerasas Dirigidas por ADN/química , AmidasRESUMEN
Nuclear magnetic relaxation is widely used to probe protein dynamics. For decades, most analyses of relaxation in proteins have relied successfully on the model-free approach, forgoing mechanistic descriptions of motion. Model-free types of correlation functions cannot describe a large carbon-13 relaxation dataset in protein side chains. Here, we use molecular dynamics simulations to design explicit models of motion and solve Fokker-Planck diffusion equations. These models of motion provide better agreement with relaxation data, mechanistic insight, and a direct link to configuration entropy.
Asunto(s)
Simulación de Dinámica Molecular , Movimiento (Física) , Difusión , EntropíaRESUMEN
Metabolomics, the systematic investigation of metabolites in biological fluids, cells, or tissues, reveals essential information about metabolism and diseases. Metabolites have functional roles in a myriad of biological processes, as substrates and products of enzymatic reactions but also as cofactors and regulators of large numbers of biochemical mechanisms. These functions involve interactions of metabolites with macromolecules. Yet, methods to systematically investigate these interactions are still scarce to date. In particular, there is a need for techniques suited to identify and characterize weak metabolite-macromolecule interactions directly in complex media such as biological fluids. Here, we introduce a method to investigate weak interactions between metabolites and macromolecules in biological fluids. Our approach is based on high-resolution NMR relaxometry and does not require any invasive procedure or separation step. We show that we can detect interactions between small and large molecules in human blood serum and quantify the size of the complex. Our work opens the way for investigations of metabolite (or other small molecules)-protein interactions in biological fluids for interactomics or pharmaceutical applications.
RESUMEN
Improving our understanding of nanosecond motions in disordered proteins requires the enhanced sampling of the spectral density function obtained from relaxation at low magnetic fields. High-resolution relaxometry and two-field NMR measurements of relaxation have, so far, only been based on the recording of one- or two-dimensional spectra, which provide insufficient resolution for challenging disordered proteins. Here, we introduce a 3D-HNCO-based two-field NMR experiment for measurements of protein backbone [Formula: see text] amide longitudinal relaxation rates. The experiment provides accurate longitudinal relaxation rates at low field (0.33 T in our case) preserving the resolution and sensitivity typical for high-field NMR spectroscopy. Radiofrequency pulses applied on six different radiofrequency channels are used to manipulate the spin system at both fields. The experiment was demonstrated on the C-terminal domain of [Formula: see text] subunit of RNA polymerase from Bacillus subtilis, a protein with highly repetitive amino-acid sequence and very low dispersion of backbone chemical shifts.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , ARN Polimerasas Dirigidas por ADN/química , Proteínas Intrínsecamente Desordenadas/química , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/químicaRESUMEN
Microtubule-associated protein 2c (MAP2c) is a 49-kDa intrinsically disordered protein regulating the dynamics of microtubules in developing neurons. MAP2c differs from its sequence homologue Tau in the pattern and kinetics of phosphorylation by cAMP-dependent protein kinase (PKA). Moreover, the mechanisms through which MAP2c interacts with its binding partners and the conformational changes and dynamics associated with these interactions remain unclear. Here, we used NMR relaxation and paramagnetic relaxation enhancement techniques to determine the dynamics and long-range interactions within MAP2c. The relaxation rates revealed large differences in flexibility of individual regions of MAP2c, with the lowest flexibility observed in the known and proposed binding sites. Quantitative conformational analyses of chemical shifts, small-angle X-ray scattering (SAXS), and paramagnetic relaxation enhancement measurements disclosed that MAP2c regions interacting with important protein partners, including Fyn tyrosine kinase, plectin, and PKA, adopt specific conformations. High populations of polyproline II and α-helices were found in Fyn- and plectin-binding sites of MAP2c, respectively. The region binding the regulatory subunit of PKA consists of two helical motifs bridged by a more extended conformation. Of note, although MAP2c and Tau did not differ substantially in their conformations in regions of high sequence identity, we found that they differ significantly in long-range interactions, dynamics, and local conformation motifs in their N-terminal domains. These results highlight that the N-terminal regions of MAP2c provide important specificity to its regulatory roles and indicate a close relationship between MAP2c's biological functions and conformational behavior.
Asunto(s)
Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Plectina/metabolismo , Conformación Proteica , Sitios de Unión , Humanos , Fosforilación , Plectina/química , Unión Proteica , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Dominios Homologos srcRESUMEN
Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.
Asunto(s)
Bacillus subtilis/enzimología , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Electricidad Estática , Secuencia de Aminoácidos , Modelos Moleculares , Conformación ProteicaRESUMEN
Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Factor sigma/metabolismo , Thermotoga maritima/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Isótopos de Carbono , Secuencia Conservada , ADN Bacteriano/química , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Isótopos de Nitrógeno , Conformación de Ácido Nucleico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factor sigma/química , Factor sigma/genética , Homología Estructural de ProteínaRESUMEN
Microtubule-associated protein 2c (MAP2c) is involved in neuronal development and is less characterized than its homolog Tau, which has various roles in neurodegeneration. Using NMR methods providing single-residue resolution and quantitative comparison, we investigated molecular interactions important for the regulatory roles of MAP2c in microtubule dynamics. We found that MAP2c and Tau significantly differ in the position and kinetics of sites that are phosphorylated by cAMP-dependent protein kinase (PKA), even in highly homologous regions. We determined the binding sites of unphosphorylated and phosphorylated MAP2c responsible for interactions with the regulatory protein 14-3-3ζ. Differences in phosphorylation and in charge distribution between MAP2c and Tau suggested that both MAP2c and Tau respond to the same signal (phosphorylation by PKA) but have different downstream effects, indicating a signaling branch point for controlling microtubule stability. Although the interactions of phosphorylated Tau with 14-3-3ζ are supposed to be a major factor in microtubule destabilization, the binding of 14-3-3ζ to MAP2c enhanced by PKA-mediated phosphorylation is likely to influence microtubule-MAP2c binding much less, in agreement with the results of our tubulin co-sedimentation measurements. The specific location of the major MAP2c phosphorylation site in a region homologous to the muscarinic receptor-binding site of Tau suggests that MAP2c also may regulate processes other than microtubule dynamics.
Asunto(s)
Proteínas 14-3-3/química , Proteínas Asociadas a Microtúbulos/química , Proteínas tau/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Microtúbulos/metabolismo , Neuronas/metabolismo , Fosforilación , Unión Proteica , Ratas , Transducción de Señal , Tubulina (Proteína)/metabolismoRESUMEN
Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg2+-bound, and beryllofluoridated CKI1RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg2+ binding and beryllofluoridation alter the conformational equilibrium of the ß3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the ß3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the ß3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the ß3-α3 loop of CKI1RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic ß3-α3 loop of CKI1RD, the corresponding loop of the ETR1 receiver domain (ETR1RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Proteínas Quinasas/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Dominios Proteicos , Proteínas Quinasas/genética , Estructura Secundaria de Proteína , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genéticaRESUMEN
Water molecules can interact with aromatic moieties using either their O-H bonds or their lone-pairs of electrons. In proteins, water-π interactions have been reported to occur with tryptophan and histidine residues, and dynamic exchange between O-Hπ hydrogen bonding and lone-pairπ interactions was suggested to take place, based on ab initio calculations. Here we used classical and QM/MM molecular dynamics simulations, complemented with an NMR study, to examine a specific water-indole interaction observed in the engrailed homeodomain and in its mutants. Our simulations indicate that the binding mode between water and indole can adapt to the potential created by the surrounding amino acids (and by the residues at the DNA surface in protein-DNA complexes), and support the model of dynamic switching between the O-Hπ hydrogen bonding and lone-pairπ binding modes.
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Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Triptófano/metabolismo , Agua/metabolismo , Animales , Drosophila , Proteínas de Drosophila , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Estructura Molecular , Mutación , Unión Proteica , Dominios Proteicos , Teoría Cuántica , Factores de Transcripción/química , Factores de Transcripción/genética , Triptófano/química , Agua/químicaRESUMEN
Hyperpolarized water in dissolution dynamic nuclear polarization (dDNP) experiments has emerged as a promising method for enhancing nuclear magnetic resonance (NMR) signals, particularly in studies of proteins and peptides. Herein, we focus on the application of "proton exchange-doubly relayed" nuclear Overhauser effects (NOE) from hyperpolarized water to achieve positive signal enhancement of methyl groups in the side chain of an alanine-glycine peptide. In particular, we show a cascade hyperpolarization transfer. Initial proton exchange between solvent and amide introduces hyperpolarization into the peptide. Subsequently, intermolecular NOE relays the hyperpolarization first to Ala-Hα and then in a second step to the Ala-CH3 moiety. Both NOEs have negative signs. Hence, the twice-relayed NOE pathway leads to a positive signal enhancement of the methyl group with respect to the thermal equilibrium magnetization. This effect might indicate a way towards hyperpolarized water-based signal enhancement for methyl groups, which are often used for NMR studies of large proteins in solution.
RESUMEN
Relaxometry consists in measuring relaxation rates over orders of magnitude of magnetic fields to probe motions of complex systems. High-resolution relaxometry (HRR) experiments can be performed on conventional high-field NMR magnets equipped with a sample shuttle. During the experiment, the sample shuttle transfers the sample between the high-field magnetic center and a chosen position in the stray field for relaxation during a variable delay, thus using the stray field as a variable field. As the relaxation delay occurs outside of the probe, HRR experiments cannot rely on the control of cross-relaxation pathways, which is standard in high-field relaxation pulse sequences. Thus, decay rates are not pure relaxation rates, which may impair a reliable description of the dynamics. Previously, we took into account cross-relaxation effects in the analysis of high-resolution relaxometry data by applying a correction factor to relaxometry decay rates in order to estimate relaxation rates. These correction factors were obtained from the iterative simulation of the relaxation decay while the sample lies outside of the probe and a preceding analysis of relaxation rates which relies on the approximation of a priori multi-exponential decays by mono-exponential functions. However, an analysis protocol matching directly experimental and simulated relaxometry decays should be more self consistent and more generally applicable as it can accommodate deviations from mono-exponential decays. Here, we introduce Matching INtensities for the Optimization of Timescales and Amplitudes of motions Under Relaxometry (MINOTAUR), a framework for the analysis of high-resolution relaxometry that takes as input the intensity decays at all fields. This approach uses the full relaxation matrix to calculate intensity decays, allowing complex relaxation pathways to be taken into account. Therefore, it eliminates the need for a correction of decay rates and for fitting multi-exponential decays with mono-exponential functions. The MINOTAUR software is designed as a flexible framework where relaxation matrices and spectral density functions corresponding to various models of motions can be defined on a case-by-case basis. The agreement with our previous analyses of protein side-chain dynamics from carbon-13 relaxation is excellent, while providing a more robust analysis tool. We expect MINOTAUR to become the tool of choice for the analysis of high-resolution relaxometry.
RESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein is an essential structural component of mature virions, encapsulating the genomic RNA and modulating RNA transcription and replication. Several of its activities might be associated with the protein's ability to undergo liquid-liquid phase separation. NSARS-CoV-2 contains an intrinsically disordered region at its N-terminus (NTE) that can be phosphorylated and is affected by mutations found in human COVID-19 infections, including in the Omicron variant of concern. Here, we show that NTE deletion decreases the range of RNA concentrations that can induce phase separation of NSARS-CoV-2 . In addition, deletion of the prion-like NTE allows NSARS-CoV-2 droplets to retain their liquid-like nature during incubation. We further demonstrate that RNA-binding engages multiple parts of the NTE and changes NTE's structural properties. The results form the foundation to characterize the impact of N-terminal mutations and post-translational modifications on the molecular properties of the SARS-CoV-2 nucleocapsid protein. STATEMENT: The nucleocapsid protein of SARS-CoV-2 plays an important role in both genome packaging and viral replication upon host infection. Replication has been associated with RNA-induced liquid-liquid phase separation of the nucleocapsid protein. We present insights into the role of the N-terminal part of the nucleocapsid protein in the protein's RNA-mediated liquid-liquid phase separation.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/genética , Humanos , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , ARN Viral/química , SARS-CoV-2/genéticaRESUMEN
Strong coupling of nuclear spins, which is achieved when their scalar coupling 2πJ is greater than or comparable to the difference Δω in their Larmor precession frequencies in an external magnetic field, gives rise to efficient coherent longitudinal polarization transfer. The strong coupling regime can be achieved when the external magnetic field is sufficiently low, as Δω is reduced proportional to the field strength. In the present work, however, we demonstrate that in heteronuclear spin systems these simple arguments may not hold, since heteronuclear spin-spin interactions alter the Δω value. The experimental method that we use is two-field nuclear magnetic resonance (NMR), exploiting sample shuttling between the high field, at which NMR spectra are acquired, and the low field, where strong couplings are expected and at which NMR pulses can be applied to affect the spin dynamics. By using this technique, we generate zero-quantum spin coherences by means of a nonadiabatic passage through a level anticrossing and study their evolution at the low field. Such zero-quantum coherences mediate the polarization transfer under strong coupling conditions. Experiments performed with a 13C-labeled amino acid clearly show that the coherent polarization transfer at the low field is pronounced in the 13C spin subsystem under proton decoupling. However, in the absence of proton decoupling, polarization transfer by coherent processes is dramatically reduced, demonstrating that heteronuclear spin-spin interactions suppress the strong coupling regime, even when the external field is low. A theoretical model is presented, which can model the reported experimental results.
RESUMEN
Merozoite surface protein 2 (MSP2) of Plasmodium falciparum is an abundant, intrinsically disordered protein that is GPI-anchored to the surface of the invasive blood stage of the malaria parasite. Recombinant MSP2 has been trialled as a component of a malaria vaccine, and is one of several disordered proteins that are candidates for inclusion in vaccines for malaria and other diseases. Nonetheless, little is known about the implications of protein disorder for the development of an effective antibody response. We have therefore undertaken a detailed analysis of the conformational dynamics of the two allelic forms of MSP2 (3D7 and FC27) using NMR spectroscopy. Chemical shifts and NMR relaxation data indicate that conformational and dynamic properties of the N- and C-terminal conserved regions in the two forms of MSP2 are essentially identical, but significant variation exists between and within the central variable regions. We observe a strong relationship between the conformational dynamics and the antigenicity of MSP2, as assessed with antisera to recombinant MSP2. Regions of increased conformational order in MSP2, including those in the conserved regions, are more strongly antigenic, while the most flexible regions are minimally antigenic. This suggests that modifications that increase conformational order may offer a means to tune the antigenicity of MSP2 and other disordered antigens, with implications for vaccine design.