Liquid-Liquid Phase Separation Modifies the Dynamic Properties of Intrinsically Disordered Proteins.

Liquid-liquid phase separation of flexible biomolecules has been identified as a ubiquitous phenomenon underlying the formation of membraneless organelles that harbor a multitude of essential cellular processes. We use nuclear magnetic resonance (NMR) spectroscopy to compare the dynamic properties of an intrinsically disordered protein (measles virus NTAIL) in the dilute and dense phases at atomic resolution. By measuring 15N NMR relaxation at different magnetic field strengths, we are able to characterize the dynamics of the protein in dilute and crowded conditions and to compare the amplitude and timescale of the different motional modes to those present in the membraneless organelle. Although the local backbone conformational sampling appears to be largely retained, dynamics occurring on all detectable timescales, including librational, backbone dihedral angle dynamics and segmental, chainlike motions, are considerably slowed down. Their relative amplitudes are also drastically modified, with slower, chain-like motions dominating the dynamic profile. In order to provide additional mechanistic insight, we performed extensive molecular dynamics simulations of the protein under self-crowding conditions at concentrations comparable to those found in the dense liquid phase. Simulation broadly reproduces the impact of formation of the condensed phase on both the free energy landscape and the kinetic interconversion between states. In particular, the experimentally observed reduction in the amplitude of the fastest component of backbone dynamics correlates with higher levels of intermolecular contacts or entanglement observed in simulations, reducing the conformational space available to this mode under strongly self-crowding conditions.

Structural insights into p300 regulation and acetylation-dependent genome organisation.

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.

Convergent views on disordered protein dynamics from NMR and computational approaches.

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.

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.

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.

The intrinsically disordered SARS-CoV-2 nucleoprotein in dynamic complex with its viral partner nsp3a.

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.

Intrinsically Disordered Tardigrade Proteins Self-Assemble into Fibrous Gels in Response to Environmental Stress.

Tardigrades are remarkable for their ability to survive harsh stress conditions as diverse as extreme temperature and desiccation. The molecular mechanisms that confer this unusual resistance to physical stress remain unknown. Recently, tardigrade-unique intrinsically disordered proteins have been shown to play an essential role in tardigrade anhydrobiosis. Here, we characterize the conformational and physical behaviour of CAHS-8 from Hypsibius exemplaris. NMR spectroscopy reveals that the protein comprises an extended central helical domain flanked by disordered termini. Upon concentration, the protein is shown to successively form oligomers, long fibres, and finally gels constituted of fibres in a strongly temperature-dependent manner. The helical domain forms the core of the fibrillar structure, with the disordered termini remaining highly dynamic within the gel. Soluble proteins can be encapsulated within cavities in the gel, maintaining their functional form. The ability to reversibly form fibrous gels may be associated with the enhanced protective properties of these proteins.

1H, 13C and 15N Backbone chemical shift assignments of the n-terminal and central intrinsically disordered domains of SARS-CoV-2 nucleoprotein.

The nucleoprotein (N) from SARS-CoV-2 is an essential cofactor of the viral replication transcription complex and as such represents an important target for viral inhibition. It has also been shown to colocalize to the transcriptase-replicase complex, where many copies of N decorate the viral genome, thereby protecting it from the host immune system. N has also been shown to phase separate upon interaction with viral RNA. N is a 419 amino acid multidomain protein, comprising two folded, RNA-binding and dimerization domains spanning residues 45-175 and 264-365 respectively. The remaining 164 amino acids are predicted to be intrinsically disordered, but there is currently no atomic resolution information describing their behaviour. Here we assign the backbone resonances of the first two intrinsically disordered domains (N1, spanning residues 1-44 and N3, spanning residues 176-263). Our assignment provides the basis for the identification of inhibitors and functional and interaction studies of this essential protein.

1H, 13C and 15N backbone chemical shift assignments of SARS-CoV-2 nsp3a.

The non-structural protein nsp3 from SARS-CoV-2 plays an essential role in the viral replication transcription complex. Nsp3a constitutes the N-terminal domain of nsp3, comprising a ubiquitin-like folded domain and a disordered acidic chain. This region of nsp3a has been linked to interactions with the viral nucleoprotein and the structure of double membrane vesicles. Here, we report the backbone resonance assignment of both domains of nsp3a. The study is carried out in the context of the international covid19-nmr consortium, which aims to characterize SARS-CoV-2 proteins and RNAs, providing for example NMR chemical shift assignments of the different viral components. Our assignment will provide the basis for the identification of inhibitors and further functional and interaction studies of this essential protein.

Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A.

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.

Measles virus nucleo- and phosphoproteins form liquid-like phase-separated compartments that promote nucleocapsid assembly.

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.

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.

Solvent-dependent segmental dynamics in intrinsically disordered proteins.

Protein and water dynamics have a synergistic relationship, which is particularly important for intrinsically disordered proteins (IDPs), although the details of this coupling remain poorly understood. Here, we combine temperature-dependent molecular dynamics simulations using different water models with extensive nuclear magnetic resonance (NMR) relaxation to examine the importance of distinct modes of solvent and solute motion for the accurate reproduction of site-specific dynamics in IDPs. We find that water dynamics play a key role in motional processes internal to "segments" of IDPs, stretches of primary sequence that share dynamic properties and behave as discrete dynamic units. We identify a relationship between the time scales of intrasegment dynamics and the lifetime of hydrogen bonds in bulk water. Correct description of these motions is essential for accurate reproduction of protein relaxation. Our findings open important perspectives for understanding the role of hydration water on the behavior and function of IDPs in solution.

Characterization of intrinsically disordered proteins and their dynamic complexes: From in vitro to cell-like environments.

Over the last two decades, it has become increasingly clear that a large fraction of the human proteome is intrinsically disordered or contains disordered segments of significant length. These intrinsically disordered proteins (IDPs) play important regulatory roles throughout biology, underlining the importance of understanding their conformational behavior and interaction mechanisms at the molecular level. Here we review recent progress in the NMR characterization of the structure and dynamics of IDPs in various functional states and environments. We describe the complementarity of different NMR parameters for quantifying the conformational propensities of IDPs in their isolated and phosphorylated states, and we discuss the challenges associated with obtaining structural models of dynamic protein-protein complexes involving IDPs. In addition, we review recent progress in understanding the conformational behavior of IDPs in cell-like environments such as in the presence of crowding agents, in membrane-less organelles and in the complex environment of the human cell.

Probing Protein Dynamics Using Multifield Variable Temperature NMR Relaxation and Molecular Dynamics Simulation.

Understanding the interplay between protein function and dynamics is currently one of the fundamental challenges of physical biology. Recently, a method using variable temperature solid-state nuclear magnetic resonance relaxation measurements has been proposed for the simultaneous measurement of 12 different activation energies reporting on distinct dynamic modes in the protein GB1. Here, we extend this approach to measure relaxation at multiple magnetic field strengths, allowing us to better constrain the motional models and to simultaneously evaluate the robustness and physical basis of the method. The data reveal backbone and side-chain motions, exhibiting low- and high-energy modes with temperature coefficients around 5 and 25 kJ·mol-1. The results are compared to variable temperature molecular dynamics simulation of the crystal lattice, providing further support for the interpretation of the experimental data in terms of molecular motion.

Conformational dynamics in crystals reveal the molecular bases for D76N beta-2 microglobulin aggregation propensity.

Spontaneous aggregation of folded and soluble native proteins in vivo is still a poorly understood process. A prototypic example is the D76N mutant of beta-2 microglobulin (ß2m) that displays an aggressive aggregation propensity. Here we investigate the dynamics of ß2m by X-ray crystallography, solid-state NMR, and molecular dynamics simulations to unveil the effects of the D76N mutation. Taken together, our data highlight the presence of minor disordered substates in crystalline ß2m. The destabilization of the outer strands of D76N ß2m accounts for the increased aggregation propensity. Furthermore, the computational modeling reveals a network of interactions with residue D76 as a keystone: this model allows predicting the stability of several point mutants. Overall, our study shows how the study of intrinsic dynamics in crystallo can provide crucial answers on protein stability and aggregation propensity. The comprehensive approach here presented may well be suited for the study of other folded amyloidogenic proteins.

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.

Atomic resolution conformational dynamics of intrinsically disordered proteins from NMR spin relaxation.

Nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful experimental approaches for investigating the conformational behaviour of intrinsically disordered proteins (IDPs). IDPs represent a significant fraction of all proteomes, and, despite their importance for understanding fundamental biological processes, the molecular basis of their activity still remains largely unknown. The functional mechanisms exploited by IDPs in their interactions with other biomolecules are defined by their intrinsic dynamic modes and associated timescales, justifying the considerable interest over recent years in the development of technologies adapted to measure and describe this behaviour. NMR spin relaxation delivers information-rich, site-specific data reporting on conformational fluctuations occurring throughout the molecule. Here we review recent progress in the use of 15N relaxation to identify local backbone dynamics and long-range chain-like motions in unfolded proteins.

Analytical Description of NMR Relaxation Highlights Correlated Dynamics in Intrinsically Disordered Proteins.

The dynamic fluctuations of intrinsically disordered proteins (IDPs) define their function. Although experimental nuclear magnetic resonance (NMR) relaxation reveals the motional complexity of these highly flexible proteins, the absence of physical models describing IDP dynamics hinders their mechanistic interpretation. Combining molecular dynamics simulation and NMR, we introduce a framework in which distinct motions are attributed to local libration, backbone dihedral angle dynamics and longer-range tumbling of one or more peptide planes. This model provides unique insight into segmental organization of dynamics in IDPs and allows us to investigate the presence and extent of the correlated motions that are essential for function.

Dynamic Descriptions of Highly Flexible Molecules from NMR Dipolar Couplings: Physical Basis and Limitations.

Biomolecules that control physiological function by changing their conformation play key roles in biology and remain poorly characterized. NMR dipolar couplings (DCs) depend intrinsically on both molecular shape and structural fluctuations, thereby providing the enticing prospect of tracking these conformational changes at atomic detail. Although this dual dependence has until now severely complicated analysis of DCs from highly dynamic systems, general approaches have recently been proposed that simplify interpretation of experimental DCs, by entirely eliminating molecular alignment from the analysis. Using simple and intuitive simulation of target ensembles, we investigate the impact of such approaches on the resulting descriptions of the conformational energy landscape. We find that ensemble descriptions of highly flexible systems derived from DCs without explicit consideration of the alignment properties of the constituent conformations can be compromised and inaccurate, despite exhibiting high correlation with experimental measurement.