Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 122
Filter
1.
Sci Adv ; 10(28): eado1453, 2024 Jul 12.
Article in English | MEDLINE | ID: mdl-38985862

ABSTRACT

The interplay between humans and their microbiome is crucial for various physiological processes, including nutrient absorption, immune defense, and maintaining homeostasis. Microbiome alterations can directly contribute to diseases or heighten their likelihood. This relationship extends beyond humans; microbiota play vital roles in other organisms, including eukaryotic pathogens causing severe diseases. Notably, Wolbachia, a bacterial microbiota, is essential for parasitic worms responsible for lymphatic filariasis and onchocerciasis, devastating human illnesses. Given the lack of rapid cures for these infections and the limitations of current treatments, new drugs are imperative. Here, we disrupt Wolbachia's symbiosis with pathogens using boron-based compounds targeting an unprecedented Wolbachia enzyme, leucyl-tRNA synthetase (LeuRS), effectively inhibiting its growth. Through a compound demonstrating anti-Wolbachia efficacy in infected cells, we use biophysical experiments and x-ray crystallography to elucidate the mechanism behind Wolbachia LeuRS inhibition. We reveal that these compounds form adenosine-based adducts inhibiting protein synthesis. Overall, our study underscores the potential of disrupting key microbiota to control infections.


Subject(s)
Microbiota , Wolbachia , Wolbachia/drug effects , Humans , Animals , Leucine-tRNA Ligase/metabolism , Leucine-tRNA Ligase/antagonists & inhibitors , Amino Acyl-tRNA Synthetases/metabolism , Amino Acyl-tRNA Synthetases/antagonists & inhibitors , Crystallography, X-Ray , Boron Compounds/pharmacology , Boron Compounds/chemistry , Symbiosis , Models, Molecular
2.
Structure ; 2024 Jul 10.
Article in English | MEDLINE | ID: mdl-39013462

ABSTRACT

The scaffold proteins JIP1 and JIP2 intervene in the c-Jun N-terminal kinase (JNK) pathway to mediate signaling specificity by coordinating the simultaneous assembly of multiple kinases. Using NMR, we demonstrate that JIP1 and JIP2 heterodimerize via their SH3 domains with the affinity of heterodimerization being comparable to homodimerization. We present the high-resolution crystal structure of the JIP2-SH3 homodimer and the JIP1-JIP2-SH3 heterodimeric complex. The JIP2-SH3 structure reveals how charge differences in residues at its dimer interface lead to formation of compensatory hydrogen bonds and salt bridges, distinguishing it from JIP1-SH3. In the JIP1-JIP2-SH3 complex, structural features of each homodimer are employed to stabilize the heterodimer. Building on these insights, we identify key residues crucial for stabilizing the dimer of both JIP1 and JIP2. Through targeted mutations in cellulo, we demonstrate a functional role for the dimerization of the JIP1 and JIP2 scaffold proteins in activation of the JNK signaling pathway.

3.
J Am Chem Soc ; 145(2): 800-810, 2023 01 18.
Article in English | MEDLINE | ID: mdl-36599057

ABSTRACT

Prodrugs have little or no pharmacological activity and are converted to active drugs in the body by enzymes, metabolic reactions, or through human-controlled actions. However, prodrugs promoting their chemical bioconversion without any of these processes have not been reported before. Here, we present an enzyme-independent prodrug activation mechanism by boron-based compounds (benzoxaboroles) targeting leucyl-tRNA synthetase (LeuRS), including an antibiotic that recently has completed phase II clinical trials to cure tuberculosis. We combine nuclear magnetic resonance spectroscopy and X-ray crystallography with isothermal titration calorimetry to show that these benzoxaboroles do not bind directly to their drug target LeuRS, instead they are prodrugs that activate their bioconversion by forming a highly specific and reversible LeuRS inhibition adduct with ATP, AMP, or the terminal adenosine of the tRNALeu. We demonstrate how the oxaborole group of the prodrugs cyclizes with the adenosine ribose at physiological concentrations to form the active molecule. This bioconversion mechanism explains the remarkably good druglike properties of benzoxaboroles showing efficacy against radically different human pathogens and fully explains the mechanism of action of these compounds. Thus, this adenosine-dependent activation mechanism represents a novel concept in prodrug chemistry that can be applied to improve the solubility, permeability and metabolic stability of challenging drugs.


Subject(s)
Amino Acyl-tRNA Synthetases , Leucine-tRNA Ligase , Prodrugs , Humans , Prodrugs/pharmacology , Adenosine/pharmacology , Leucine-tRNA Ligase/genetics , Anti-Bacterial Agents/pharmacology
4.
Cell Rep Med ; 3(4): 100582, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35492241

ABSTRACT

The gastric hormone ghrelin stimulates food intake and increases plasma glucose through activation of the growth hormone secretagogue receptor (GHSR). Liver-expressed antimicrobial peptide 2 (LEAP2) has been proposed to inhibit actions of ghrelin through inverse effects on GHSR activity. Here, we investigate the effects of exogenous LEAP2 on postprandial glucose metabolism and ad libitum food intake in a randomized, double-blind, placebo-controlled, crossover trial of 20 healthy men. We report that LEAP2 infusion lowers postprandial plasma glucose and growth hormone concentrations and decreases food intake during an ad libitum meal test. In wild-type mice, plasma glucose and food intake are reduced by LEAP2 dosing, but not in GHSR-null mice, pointing to GHSR as a potential mediator of LEAP2's glucoregulatory and appetite-suppressing effects in mice.


Subject(s)
Antimicrobial Cationic Peptides/therapeutic use , Ghrelin , Glucose , Animals , Blood Glucose , Eating , Glucose/pharmacology , Humans , Mice , Receptors, Ghrelin
5.
Chem Rev ; 122(10): 9331-9356, 2022 05 25.
Article in English | MEDLINE | ID: mdl-35446534

ABSTRACT

Intrinsically disordered proteins are ubiquitous throughout all known proteomes, playing essential roles in all aspects of cellular and extracellular biochemistry. To understand their function, it is necessary to determine their structural and dynamic behavior and to describe the physical chemistry of their interaction trajectories. Nuclear magnetic resonance is perfectly adapted to this task, providing ensemble averaged structural and dynamic parameters that report on each assigned resonance in the molecule, unveiling otherwise inaccessible insight into the reaction kinetics and thermodynamics that are essential for function. In this review, we describe recent applications of NMR-based approaches to understanding the conformational energy landscape, the nature and time scales of local and long-range dynamics and how they depend on the environment, even in the cell. Finally, we illustrate the ability of NMR to uncover the mechanistic basis of functional disordered molecular assemblies that are important for human health.


Subject(s)
Intrinsically Disordered Proteins , Humans , Intrinsically Disordered Proteins/chemistry , Magnetic Resonance Spectroscopy , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Thermodynamics
7.
J Mol Biol ; 434(10): 167551, 2022 05 30.
Article in English | MEDLINE | ID: mdl-35317998

ABSTRACT

To understand the dynamic interactions between the phosphoprotein (P) and the nucleoprotein (N) within the transcription/replication complex of the Paramyxoviridae and to decipher their roles in regulating viral multiplication, we characterized the structural properties of the C-terminal X domain (PXD) of Nipah (NiV) and Hendra virus (HeV) P protein. In crystals, isolated NiV PXD adopted a two-helix dimeric conformation, which was incompetent for binding its partners, but in complex with the C-terminal intrinsically disordered tail of the N protein (NTAIL), it folded into a canonical 3H bundle conformation. In solution, SEC-MALLS, SAXS and NMR spectroscopy experiments indicated that both NiV and HeV PXD were larger in size than expected for compact proteins of the same molecular mass and were in conformational exchange between a compact three-helix (3H) bundle and partially unfolded conformations, where helix α3 is detached from the other two. Some measurements also provided strong evidence for dimerization of NiV PXD in solution but not for HeV PXD. Ensemble modeling of experimental SAXS data and statistical-dynamical modeling reconciled all these data, yielding a model where NiV and HeV PXD exchanged between different conformations, and where NiV but not HeV PXD formed dimers. Finally, recombinant NiV comprising a chimeric P carrying HeV PXD was rescued and compared with parental NiV. Experiments carried out in cellula demonstrated that the replacement of PXD did not significantly affect the replication dynamics while caused a slight virus attenuation, suggesting a possible role of the dimerization of NiV PXD in viral replication.


Subject(s)
Hendra Virus , Nipah Virus , Nucleocapsid Proteins , Phosphoproteins , Viral Proteins , Virus Replication , Hendra Virus/genetics , Hendra Virus/physiology , Humans , Nipah Virus/genetics , Nipah Virus/physiology , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Domains , Protein Folding , Protein Multimerization , Scattering, Small Angle , Viral Proteins/chemistry , Viral Proteins/genetics , X-Ray Diffraction
8.
Nature ; 602(7898): 695-700, 2022 02.
Article in English | MEDLINE | ID: mdl-35173330

ABSTRACT

Aromatic residues cluster in the core of folded proteins, where they stabilize the structure through multiple interactions. Nuclear magnetic resonance (NMR) studies in the 1970s showed that aromatic side chains can undergo ring flips-that is, 180° rotations-despite their role in maintaining the protein fold1-3. It was suggested that large-scale 'breathing' motions of the surrounding protein environment would be necessary to accommodate these ring flipping events1. However, the structural details of these motions have remained unclear. Here we uncover the structural rearrangements that accompany ring flipping of a buried tyrosine residue in an SH3 domain. Using NMR, we show that the tyrosine side chain flips to a low-populated, minor state and, through a proteome-wide sequence analysis, we design mutants that stabilize this state, which allows us to capture its high-resolution structure by X-ray crystallography. A void volume is generated around the tyrosine ring during the structural transition between the major and minor state, and this allows fast flipping to take place. Our results provide structural insights into the protein breathing motions that are associated with ring flipping. More generally, our study has implications for protein design and structure prediction by showing how the local protein environment influences amino acid side chain conformations and vice versa.


Subject(s)
Proteins , Tyrosine , Crystallography, X-Ray , Motion , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Tyrosine/chemistry , Tyrosine/metabolism , src Homology Domains
9.
Sci Adv ; 8(3): eabm4034, 2022 Jan 21.
Article in English | MEDLINE | ID: mdl-35044811

ABSTRACT

The processes of genome replication and transcription of SARS-CoV-2 represent important targets for viral inhibition. Betacoronaviral nucleoprotein (N) is a highly dynamic cofactor of the replication-transcription complex (RTC), whose function depends on an essential interaction with the amino-terminal ubiquitin-like domain of nsp3 (Ubl1). Here, we describe this complex (dissociation constant - 30 to 200 nM) at atomic resolution. The interaction implicates two linear motifs in the intrinsically disordered linker domain (N3), a hydrophobic helix (219LALLLLDRLNQL230) and a disordered polar strand (243GQTVTKKSAAEAS255), that mutually engage to form a bipartite interaction, folding N3 around Ubl1. This results in substantial collapse in the dimensions of dimeric N, forming a highly compact molecular chaperone, that regulates binding to RNA, suggesting a key role of nsp3 in the association of N to the RTC. The identification of distinct linear motifs that mediate an important interaction between essential viral factors provides future targets for development of innovative strategies against COVID-19.

10.
J Am Chem Soc ; 143(48): 20109-20121, 2021 12 08.
Article in English | MEDLINE | ID: mdl-34817999

ABSTRACT

Studying the conformational landscape of intrinsically disordered and partially folded proteins is challenging and only accessible to a few solution state techniques, such as nuclear magnetic resonance (NMR), small-angle scattering techniques, and single-molecule Förster resonance energy transfer (smFRET). While each of the techniques is sensitive to different properties of the disordered chain, such as local structural propensities, overall dimension, or intermediate- and long-range contacts, conformational ensembles describing intrinsically disordered proteins (IDPs) accurately should ideally respect all of these properties. Here we develop an integrated approach using a large set of FRET efficiencies and fluorescence lifetimes, NMR chemical shifts, and paramagnetic relaxation enhancements (PREs), as well as small-angle X-ray scattering (SAXS) to derive quantitative conformational ensembles in agreement with all parameters. Our approach is tested using simulated data (five sets of PREs and 15 FRET efficiencies) and validated experimentally on the example of the disordered domain of measles virus phosphoprotein, providing new insights into the conformational landscape of this viral protein that comprises transient structural elements and is more compact than an unfolded chain throughout its length. Rigorous cross-validation using FRET efficiencies, fluorescence lifetimes, and SAXS demonstrates the predictive nature of the calculated conformational ensembles and underlines the potential of this strategy in integrative dynamic structural biology.


Subject(s)
Intrinsically Disordered Proteins/chemistry , Algorithms , Fluorescence Resonance Energy Transfer , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Scattering, Small Angle , X-Ray Diffraction
11.
Int Emerg Nurs ; 58: 101054, 2021 Sep.
Article in English | MEDLINE | ID: mdl-34530314

ABSTRACT

INTRODUCTION: Chronic obstructive pulmonary disease causes significant burdens for patients. To secure safe care and treatment in the first part of the patient trajectory, it is essential to explore patients' experiences during admission in the acute phase following an exacerbation due to COPD. METHODS: Thirteen semi-structured interviews were performed, using Kvale and Brinkman's meaning condensation as an analytical approach. RESULTS: The findings are illustrated in four themes: A burdened patient group; the urgent need for air; to be in a cross field between chaos, waiting time and uncertainty; and communication as the lifeline. CONCLUSION: Patients were distressed due to shortness of breath and anxiety in the busy setting of the emergency department, and the analysis showed that breathing was the most important and urgent issue when the patients were acutely admitted. This study contributes to the evidence base on how the first part of the trajectory affects patients' ability to master their situation and their overall well-being.


Subject(s)
Pulmonary Disease, Chronic Obstructive , Dyspnea/etiology , Emergency Service, Hospital , Hospitalization , Humans , Pulmonary Disease, Chronic Obstructive/complications , Qualitative Research
12.
Biomolecules ; 11(8)2021 08 13.
Article in English | MEDLINE | ID: mdl-34439869

ABSTRACT

Intrinsically disordered proteins (IDPs) can engage in promiscuous interactions with their protein targets; however, it is not clear how this feature is encoded in the primary sequence of the IDPs and to what extent the surface properties and the shape of the binding cavity dictate the binding mode and the final bound conformation. Here we show, using a combination of nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), that the promiscuous interaction of the intrinsically disordered regulatory domain of the mitogen-activated protein kinase kinase MKK4 with p38α and JNK1 is facilitated by folding-upon-binding into two different conformations, despite the high sequence conservation and structural homology between p38α and JNK1. Our results support a model whereby the specific surface properties of JNK1 and p38α dictate the bound conformation of MKK4 and that enthalpy-entropy compensation plays a major role in maintaining comparable binding affinities for MKK4 towards the two kinases.


Subject(s)
JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase 14 , Models, Molecular , Humans , JNK Mitogen-Activated Protein Kinases/chemistry , JNK Mitogen-Activated Protein Kinases/metabolism , Kinetics , MAP Kinase Kinase 4/chemistry , MAP Kinase Kinase 4/metabolism , Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/metabolism , Protein Binding , Protein Conformation , Protein Folding
13.
Nucleic Acids Res ; 49(D1): D404-D411, 2021 01 08.
Article in English | MEDLINE | ID: mdl-33305318

ABSTRACT

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.


Subject(s)
Databases, Protein , Intrinsically Disordered Proteins/chemistry , Humans , Search Engine , Tumor Suppressor Protein p53/chemistry
14.
Proc Natl Acad Sci U S A ; 117(34): 20576-20585, 2020 08 25.
Article in English | MEDLINE | ID: mdl-32788352

ABSTRACT

Temperate bacteriophages can enter one of two life cycles following infection of a sensitive host: the lysogenic or the lytic life cycle. The choice between the two alternative life cycles is dependent upon a tight regulation of promoters and their cognate regulatory proteins within the phage genome. We investigated the genetic switch of TP901-1, a bacteriophage of Lactococcus lactis, controlled by the CI repressor and the modulator of repression (MOR) antirepressor and their interactions with DNA. We determined the solution structure of MOR, and we solved the crystal structure of MOR in complex with the N-terminal domain of CI, revealing the structural basis of MOR inhibition of CI binding to the DNA operator sites. 15N NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion and rotating frame R1ρ measurements demonstrate that MOR displays molecular recognition dynamics on two different time scales involving a repacking of aromatic residues at the interface with CI. Mutations in the CI:MOR binding interface impair complex formation in vitro, and when introduced in vivo, the bacteriophage switch is unable to choose the lytic life cycle showing that the CI:MOR complex is essential for proper functioning of the genetic switch. On the basis of sequence alignments, we show that the structural features of the MOR:CI complex are likely conserved among a larger family of bacteriophages from human pathogens implicated in transfer of antibiotic resistance.


Subject(s)
Bacteriophages/physiology , Lysogeny , Repressor Proteins/physiology , Viral Regulatory and Accessory Proteins/physiology , Genome, Bacterial , Host-Pathogen Interactions , Kinetics , Lactococcus lactis/virology , Molecular Dynamics Simulation , Operator Regions, Genetic , Protein Conformation , Repressor Proteins/chemistry , Viral Regulatory and Accessory Proteins/chemistry
15.
Nat Commun ; 11(1): 3656, 2020 07 21.
Article in English | MEDLINE | ID: mdl-32694517

ABSTRACT

Avian influenza polymerase undergoes host adaptation in order to efficiently replicate in human cells. Adaptive mutants are localised on the C-terminal (627-NLS) domains of the PB2 subunit. In particular, mutation of PB2 residue 627 from E to K rescues polymerase activity in mammalian cells. A host transcription regulator ANP32A, comprising a long C-terminal intrinsically disordered domain (IDD), is responsible for this adaptation. Human ANP32A IDD lacks a 33 residue insertion compared to avian ANP32A, and this deletion restricts avian influenza polymerase activity. We used NMR to determine conformational ensembles of E627 and K627 forms of 627-NLS of PB2 in complex with avian and human ANP32A. Human ANP32A IDD transiently binds to the 627 domain, exploiting multivalency to maximise affinity. E627 interrupts the polyvalency of the interaction, an effect compensated by an avian-unique motif in the IDD. The observed binding mode is maintained in the context of heterotrimeric influenza polymerase, placing ANP32A in the immediate vicinity of known host-adaptive PB2 mutants.


Subject(s)
Avian Proteins/ultrastructure , Influenza A Virus, H5N1 Subtype/pathogenicity , Nuclear Proteins/ultrastructure , Protein Domains/genetics , RNA-Binding Proteins/ultrastructure , RNA-Dependent RNA Polymerase/ultrastructure , Viral Proteins/ultrastructure , Animals , Avian Proteins/metabolism , Birds/virology , Humans , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/metabolism , Influenza in Birds/virology , Influenza, Human/virology , Mutation , Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/metabolism , Protein Binding/genetics , RNA-Binding Proteins/metabolism , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , Species Specificity , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication
16.
Curr Opin Virol ; 41: 59-67, 2020 04.
Article in English | MEDLINE | ID: mdl-32570195

ABSTRACT

The measles virus replication complex represents a potentially important, but as yet relatively unexplored target for viral inhibition. Little is known about the molecular mechanisms that underpin replication and transcription in paramyxoviruses. In recent years it has become clear that conformational dynamics play an important role in paramyxoviral replication, and that a complete understanding of the viral cycle requires a description of the structural plasticity of the different components. Here, we review recent progress in this direction, covering the dynamics of the nucleocapsid assembly process, high resolution structure and dynamics of protein:RNA interactions, and the investigation of the role of intrinsic conformational disorder in pre-assembly nucleoprotein/phosphoprotein complexes. Finally, we discuss the role of viral factories in the form of phase-separated membraneless organelles formed by measles virus phospho and nucleoproteins that promote the assembly of nucleocapsid structures.


Subject(s)
Measles virus/physiology , Measles/virology , Nucleocapsid/chemistry , RNA, Viral/genetics , Virus Replication , Animals , Humans , Measles virus/chemistry , Measles virus/genetics , Nucleocapsid/genetics , Nucleocapsid/metabolism , Nucleoproteins/chemistry , Nucleoproteins/genetics , Nucleoproteins/metabolism , RNA, Viral/chemistry , RNA, Viral/metabolism
17.
Biophys J ; 118(10): 2470-2488, 2020 05 19.
Article in English | MEDLINE | ID: mdl-32348724

ABSTRACT

The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah virus (NiV) phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoviruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.


Subject(s)
Nipah Virus , Phosphoproteins , Protein Conformation , Viral Proteins , Virus Replication
18.
Sci Adv ; 6(14): eaaz7095, 2020 04.
Article in English | MEDLINE | ID: mdl-32270045

ABSTRACT

Many viruses are known to form cellular compartments, also called viral factories. Paramyxoviruses, including measles virus, colocalize their proteomic and genomic material in puncta in infected cells. We demonstrate that purified nucleoproteins (N) and phosphoproteins (P) of measles virus form liquid-like membraneless organelles upon mixing in vitro. We identify weak interactions involving intrinsically disordered domains of N and P that are implicated in this process, one of which is essential for phase separation. Fluorescence allows us to follow the modulation of the dynamics of N and P upon droplet formation, while NMR is used to investigate the thermodynamics of this process. RNA colocalizes to droplets, where it triggers assembly of N protomers into nucleocapsid-like particles that encapsidate the RNA. The rate of encapsidation within droplets is enhanced compared to the dilute phase, revealing one of the roles of liquid-liquid phase separation in measles virus replication.


Subject(s)
Measles virus/physiology , Nucleocapsid/metabolism , Nucleoproteins/metabolism , Phosphoproteins/metabolism , Viral Proteins/metabolism , Virus Assembly , Magnetic Resonance Spectroscopy , Measles/virology , Nucleoproteins/chemistry , Phosphoproteins/chemistry , Protein Binding , Protein Interaction Domains and Motifs , RNA, Viral , Recombinant Proteins , Thermodynamics , Virus Replication
19.
J Am Chem Soc ; 141(44): 17817-17829, 2019 11 06.
Article in English | MEDLINE | ID: mdl-31591893

ABSTRACT

Intrinsically disordered proteins (IDPs) are flexible biomolecules whose essential functions are defined by their dynamic nature. Nuclear magnetic resonance (NMR) spectroscopy is ideally suited to the investigation of this behavior at atomic resolution. NMR relaxation is increasingly used to detect conformational dynamics in free and bound forms of IDPs under conditions approaching physiological, although a general framework providing a quantitative interpretation of these exquisitely sensitive probes as a function of experimental conditions is still lacking. Here, measuring an extensive set of relaxation rates sampling multiple-time-scale dynamics over a broad range of crowding conditions, we develop and test an integrated analytical description that accurately portrays the motion of IDPs as a function of the intrinsic properties of the crowded molecular environment. In particular we observe a strong dependence of both short-range and long-range motional time scales of the protein on the friction of the solvent. This tight coupling between the dynamic behavior of the IDP and its environment allows us to develop analytical expressions for protein motions and NMR relaxation properties that can be accurately applied over a vast range of experimental conditions. This unified dynamic description provides new insight into the physical behavior of IDPs, extending our ability to quantitatively investigate their conformational dynamics under complex environmental conditions, and accurately predicting relaxation rates reporting on motions on time scales up to tens of nanoseconds, both in vitro and in cellulo.


Subject(s)
Intrinsically Disordered Proteins/chemistry , MAP Kinase Kinase 4/chemistry , Nucleoproteins/chemistry , Viral Proteins/chemistry , Animals , Nitrogen Isotopes/chemistry , Nuclear Magnetic Resonance, Biomolecular , Oocytes/chemistry , Protein Conformation , Protein Domains , Sendai virus/chemistry , Xenopus laevis
20.
J Am Chem Soc ; 141(42): 16817-16828, 2019 10 23.
Article in English | MEDLINE | ID: mdl-31550880

ABSTRACT

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.


Subject(s)
Bacillus subtilis/enzymology , DNA-Directed RNA Polymerases/chemistry , DNA-Directed RNA Polymerases/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolism , Static Electricity , Amino Acid Sequence , Models, Molecular , Protein Conformation
SELECTION OF CITATIONS
SEARCH DETAIL
...