Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

Visualizing protein breathing motions associated with aromatic ring flipping.

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.

NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins.

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.

Adenosine-Dependent Activation Mechanism of Prodrugs Targeting an Aminoacyl-tRNA Synthetase.

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.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

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.

Revealing the mechanism of repressor inactivation during switching of a temperate bacteriophage.

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.

Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication.

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5') in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5' and 3' binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3' end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

Quantitative Description of Intrinsically Disordered Proteins Using Single-Molecule FRET, NMR, and SAXS.

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.

Structural Description of the Nipah Virus Phosphoprotein and Its Interaction with STAT1.

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.

Self-association of a highly charged arginine-rich cell-penetrating peptide.

Small-angle X-ray scattering (SAXS) measurements reveal a striking difference in intermolecular interactions between two short highly charged peptides-deca-arginine (R10) and deca-lysine (K10). Comparison of SAXS curves at high and low salt concentration shows that R10 self-associates, while interactions between K10 chains are purely repulsive. The self-association of R10 is stronger at lower ionic strengths, indicating that the attraction between R10 molecules has an important electrostatic component. SAXS data are complemented by NMR measurements and potentials of mean force between the peptides, calculated by means of umbrella-sampling molecular dynamics (MD) simulations. All-atom MD simulations elucidate the origin of the R10-R10 attraction by providing structural information on the dimeric state. The last two C-terminal residues of R10 constitute an adhesive patch formed by stacking of the side chains of two arginine residues and by salt bridges formed between the like-charge ion pair and the C-terminal carboxyl groups. A statistical analysis of the Protein Data Bank reveals that this mode of interaction is a common feature in proteins.

A Unified Description of Intrinsically Disordered Protein Dynamics under Physiological Conditions Using NMR Spectroscopy.

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.

Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase.

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.

Deciphering the Dynamic Interaction Profile of an Intrinsically Disordered Protein by NMR Exchange Spectroscopy.

Intrinsically disordered proteins (IDPs) display a large number of interaction modes including folding-upon-binding, binding without major structural transitions, or binding through highly dynamic, so-called fuzzy, complexes. The vast majority of experimental information about IDP binding modes have been inferred from crystal structures of proteins in complex with short peptides of IDPs. However, crystal structures provide a mainly static view of the complexes and do not give information about the conformational dynamics experienced by the IDP in the bound state. Knowledge of the dynamics of IDP complexes is of fundamental importance to understand how IDPs engage in highly specific interactions without concomitantly high binding affinity. Here, we combine rotating-frame R1ρ, Carr-Purcell-Meiboom Gill relaxation dispersion as well as chemical exchange saturation transfer to decipher the dynamic interaction profile of an IDP in complex with its partner. We apply the approach to the dynamic signaling complex formed between the mitogen-activated protein kinase (MAPK) p38α and the intrinsically disordered regulatory domain of the MAPK kinase MKK4. Our study demonstrates that MKK4 employs a subtle combination of interaction modes in order to bind to p38α, leading to a complex displaying significantly different dynamics across the bound regions.

Structure and dynamics of the MKK7-JNK signaling complex.

Signaling specificity in the mitogen-activated protein kinase (MAPK) pathways is controlled by disordered domains of the MAPK kinases (MKKs) that specifically bind to their cognate MAPKs via linear docking motifs. MKK7 activates the c-Jun N-terminal kinase (JNK) pathway and is the only MKK containing three motifs within its regulatory domain. Here, we characterize the conformational behavior and interaction mechanism of the MKK7 regulatory domain. Using NMR spectroscopy, we develop an atomic resolution ensemble description of MKK7, revealing highly diverse intrinsic conformational propensities of the three docking sites, suggesting that prerecognition sampling of the bound-state conformation is not prerequisite for binding. Although the different sites exhibit similar affinities for JNK1, interaction kinetics differ considerably. Importantly, we determine the crystal structure of JNK1 in complex with the second docking site of MKK7, revealing two different binding modes of the docking motif correlating with observations from NMR exchange spectroscopy. Our results provide unique insight into how signaling specificity is regulated by linear motifs and, in general, into the role of conformational disorder in MAPK signaling.

A Combined NMR and SAXS Analysis of the Partially Folded Cataract-Associated V75D γD-Crystallin.

A cataract is a pathological condition characterized by the clouding of the normally clear eye lens brought about by deposition of crystallin proteins in the lens fiber cells. These protein aggregates reduce visual acuity by scattering or blocking incoming light. Chemical damage to proteins of the crystallin family, accumulated over a lifetime, leads to age-related cataract, whereas inherited mutations are associated with congenital or early-onset cataract. The V75D mutant of γD-crystallin is associated with congenital cataract in mice and was previously shown to un/fold via a partially folded intermediate. Here, we structurally characterized the stable equilibrium urea unfolding intermediate of V75D at the ensemble level using solution NMR and small-angle x-ray scattering. Our data show that, in the intermediate, the C-terminal domain retains a folded conformation that is similar to the native wild-type protein, whereas the N-terminal domain is unfolded and comprises an ensemble of random conformers, without any detectable residual structural propensities.

The LC8 Recognition Motif Preferentially Samples Polyproline II Structure in Its Free State.

LC8 is a ubiquitous hub protein that binds intrinsically disordered proteins and promotes their assembly into higher-order complexes. A common feature among the more than 100 essential LC8 binding proteins is that in the 10-12-amino acid recognition sequence there is a conserved QT motif but variable amino acids N- and C-terminal to the QT pair. The sequence diversity among LC8 binding partners implies that structural factors also contribute to specificity. To investigate whether one such factor is the transient secondary structure favored by an LC8 binding sequence, we report here a molecular ensemble description of ICTL, a domain of the dynein intermediate chain that includes binding sites for light chains LC8 and Tctex1. Nuclear magnetic resonance secondary chemical shifts and residual dipolar coupling values combined with ensemble generation and selection algorithms indicate a deviation from statistical (random) coil behavior with an elevated population of polyproline II (PPII) conformations for the ICTL regions that bind LC8 and Tctex1. Independent measurements of one- and three-bond scalar couplings confirm the PPII transient secondary structure propensity. Given that in the IC/Tctex1/LC8 ternary complex ICTL forms a ß-strand at the interface of Tctex1 and LC8, we hypothesize that a PPII conformation may facilitate its initial docking and insertion into the binding cleft of the ß-sheet LC8 dimer interface. Molecular ensemble calculations for intrinsically disordered LC8 binding partners also reveal PPII conformational sampling within and proximate to the LC8 recognition motifs, suggesting that a preference for a PPII conformation is general for LC8 binding partners.

Identification of Dynamic Modes in an Intrinsically Disordered Protein Using Temperature-Dependent NMR Relaxation.

The dynamic modes and time scales sampled by intrinsically disordered proteins (IDPs) define their function. Nuclear magnetic resonance (NMR) spin relaxation is probably the most powerful tool for investigating these motions delivering site-specific descriptions of conformational fluctuations from throughout the molecule. Despite the abundance of experimental measurement of relaxation in IDPs, the physical origin of the measured relaxation rates remains poorly understood. Here we measure an extensive range of auto- and cross-correlated spin relaxation rates at multiple magnetic field strengths on the C-terminal domain of the nucleoprotein of Sendai virus, over a large range of temperatures (268-298 K), and combine these data to describe the dynamic behavior of this archetypal IDP. An Arrhenius-type relationship is used to simultaneously analyze up to 61 relaxation rates per amino acid over the entire temperature range, allowing the measurement of local activation energies along the chain, and the assignment of physically distinct dynamic modes. Fast (τ ≤ 50 ps) components report on librational motions, a dominant mode occurs on time scales around 1 ns, apparently reporting on backbone sampling within Ramachandran substates, while a slower component (5-25 ns) reports on segmental dynamics dominated by the chain-like nature of the protein. Extending the study to three protein constructs of different lengths (59, 81, and 124 amino acids) substantiates the assignment of these contributions. The analysis is shown to be remarkably robust, accurately predicting a broad range of relaxation data measured at different magnetic field strengths and temperatures. The ability to delineate intrinsic modes and time scales from NMR spin relaxation will improve our understanding of the behavior and function of IDPs, adding a new and essential dimension to the description of this biologically important and ubiquitous class of proteins.

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, has a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We define a new mechanism of allosteric inhibition that targets the C-terminal, noncatalytic segment of PTP1B. We present what is to our knowledge the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small-molecule inhibitor MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence.

Measles virus RNA genomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleo-proteins (N) that bind the entire genome. N-RNA provides the template for replication and transcription by the viral polymerase and is a promising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs. Here, we demonstrate self-organization of N into NC-like particles in vitro upon addition of RNA, providing a simple and versatile tool for investigating assembly. Real-time NMR and fluorescence spectroscopy reveals biphasic assembly kinetics. Remarkably, assembly depends strongly on the RNA-sequence, with the genomic 5' end and poly-Adenine sequences assembling efficiently, while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

Structural and biophysical characterization of murine rif1 C terminus reveals high specificity for DNA cruciform structures.

Mammalian Rif1 is a key regulator of DNA replication timing, double-stranded DNA break repair, and replication fork restart. Dissecting the molecular functions of Rif1 is essential to understand how it regulates such diverse processes. However, Rif1 is a large protein that lacks well defined functional domains and is predicted to be largely intrinsically disordered; these features have hampered recombinant expression of Rif1 and subsequent functional characterization. Here we applied ESPRIT (expression of soluble proteins by random incremental truncation), an in vitro evolution-like approach, to identify high yielding soluble fragments encompassing conserved regions I and II (CRI and CRII) at the C-terminal region of murine Rif1. NMR analysis showed CRI to be intrinsically disordered, whereas CRII is partially folded. CRII binds cruciform DNA with high selectivity and micromolar affinity and thus represents a functional DNA binding domain. Mutational analysis revealed an α-helical region of CRII to be important for cruciform DNA binding and identified critical residues. Thus, we present the first structural study of the mammalian Rif1, identifying a domain that directly links its function to DNA binding. The high specificity of Rif1 for cruciform structures is significant given the role of this key protein in regulating origin firing and DNA repair.