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.

Multivalent Dynamic Colocalization of Avian Influenza Polymerase and Nucleoprotein by Intrinsically Disordered ANP32A Reveals the Molecular Basis of Human Adaptation.

Adaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remains poorly understood. Here we characterize ternary complexes formed between ANP32A, FluPol, and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted FluPol, containing the E627 K mutation, simultaneously bind the identical extended linear motif on human ANP32A in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.

Detection of Metabolite-Protein Interactions in Complex Biological Samples by High-Resolution Relaxometry: Toward Interactomics by NMR.

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.

Hsp70 biases the folding pathways of client proteins.

The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a (1)H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.

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.

Dramatic Decrease in CEST Measurement Times Using Multi-Site Excitation.

Chemical exchange saturation transfer (CEST) has recently evolved into a powerful approach for studying sparsely populated, "invisible" protein states in slow exchange with a major, visible conformer. Central to the technique is the use of a weak, highly selective radio-frequency field that is applied at different frequency offsets in successive experiments, "searching" for minor state resonances. The recording of CEST profiles with enough points to ensure coverage of the entire spectrum at sufficient resolution can be time-consuming, especially for applications that require high static magnetic fields or when small chemical shift differences between exchanging states must be quantified. Here, we show - with applications involving 15 N CEST - that the process can be significantly accelerated by using a multi-frequency irradiation scheme, leading in some applications to an order of magnitude savings in measurement time.

Mapping the conformation of a client protein through the Hsp70 functional cycle.

The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP- and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaK-bound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.

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.

Structure and Dynamics of an Intrinsically Disordered Protein Region That Partially Folds upon Binding by Chemical-Exchange NMR.

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant.

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein's structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the 'invisible', excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein's function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

Measuring hydrogen exchange rates in invisible protein excited states.

Hydrogen exchange rates have become a valuable probe for studying the relationship between dynamics and structure and for dissecting the mechanism by which proteins fold to their native conformation. Typically measured rates correspond to averages over all protein states from which hydrogen exchange can occur. Here we describe a new NMR experiment based on chemical exchange saturation transfer that provides an avenue for obtaining uncontaminated, per-residue amide hydrogen exchange rates for interconverting native and invisible states so long as they can be separated on the basis of distinct (15)N chemical shifts. The approach is applied to the folding reaction of the Fyn SH3 domain that exchanges between a highly populated, NMR-visible native state and a conformationally excited, NMR-invisible state, corresponding to the unfolded ensemble. Excellent agreement between experimentally derived hydrogen exchange rates of the excited state at a pair of pHs is obtained, taking into account the expected dependence of exchange on pH. Extracted rates for the unfolded ensemble have been used to test hydrogen exchange predictions based on the primary protein sequence that are used in many analyses of solvent exchange rates, with a Pearson correlation coefficient of 0.84 obtained.

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Structural biology turned on its head.

NMR spectroscopy and ITC have recently been combined to demonstrate how phosphorylation of the intrinsically disordered eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) induces folding into a stable three-dimensional structure. The classical structure-function paradigm is inverted, with phosphorylation-induced folding inhibiting binding to the eukaryotic translation initiation factor 4E (eIF4E) and thereby contributing to regulation of the interaction.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.

Probing slowly exchanging protein systems via ¹³Cα-CEST: monitoring folding of the Im7 protein.

A¹³C(α) chemical exchange saturation transfer based experiment is presented for the study of protein systems undergoing slow interconversion between an 'observable' ground state and one or more 'invisible' excited states. Here a labeling strategy whereby [2-(13)C]-glucose is the sole carbon source is exploited, producing proteins with ¹³C at the C(α) position, while the majority of residues remain unlabeled at CO or C(ß). The new experiment is demonstrated with an application to the folding reaction of the Im7 protein that involves an on-pathway excited state. The obtained excited state (13)C(α) chemical shifts are cross validated by comparison to values extracted from analysis of CPMG relaxation dispersion profiles, establishing the utility of the methodology.

A computational study of the effects of (13) C-(13) C scalar couplings on (13) C CEST NMR spectra: towards studies on a uniformly (13) C-labeled protein.

Read the label: The NMR CEST experiment can be used to reconstruct spectra of sparsely populated, transiently formed protein conformers so long as they exchange with a highly populated ground state with rates of 20-300 s(-1) . Here we establish that accurate (13) C chemical shifts of side-chain carbon nuclei can be obtained from uniformly (13) C-labeled samples, without interference from the coupled (13) C spin network.

Comprehensive analysis of relaxation decays from high-resolution relaxometry.

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.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here, we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at atomic resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs led to the formation of XLF and X4L4 condensates in vitro which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome editing strategies.

Studying "invisible" excited protein states in slow exchange with a major state conformation.

Ever since its initial development, solution NMR spectroscopy has been used as a tool to study conformational exchange. Although many systems are amenable to relaxation dispersion approaches, cases involving highly skewed populations in slow chemical exchange have, in general, remained recalcitrant to study. Here an experiment to detect and characterize "invisible" excited protein states in slow exchange with a visible ground-state conformation (excited-state lifetimes ranging from ∼5 to 50 ms) is presented. This method, which is an adaptation of the chemical exchange saturation transfer (CEST) magnetic resonance imaging experiment, involves irradiating various regions of the spectrum with a weak B(1) field while monitoring the effect on the visible major-state peaks. The variation in major-state peak intensities as a function of frequency offset and B(1) field strength is quantified to obtain the minor-state population, its lifetime, and excited-state chemical shifts and line widths. The methodology was validated with (15)N CEST experiments recorded on an SH3 domain-ligand exchanging system and subsequently used to study the folding transition of the A39G FF domain, where the invisible unfolded state has a lifetime of ∼20 ms. Far more accurate exchange parameters and chemical shifts were obtained than via analysis of Carr-Purcell-Meiboom-Gill relaxation dispersion data.

A 2D ¹³C-CEST experiment for studying slowly exchanging protein systems using methyl probes: an application to protein folding.

A 2D (13)C Chemical Exchange Saturation Transfer (CEST) experiment is presented for studying slowly exchanging protein systems using methyl groups as probes. The utility of the method is first established through studies of protein L, a small protein, for which chemical exchange on the millisecond time-scale is not observed. Subsequently the approach is applied to a folding exchange reaction of a G48M mutant Fyn SH3 domain, for which only cross-peaks derived from the folded ('ground') state are present in spectra. Fits of (15)N and methyl (13)C CEST profiles of the Fyn SH3 domain establish that the exchange reaction involves an interchange between folded and unfolded conformers, although elevated methyl (13)C transverse relaxation rates for some of the residues of the unfolded ('invisible, excited') state indicate that it likely exchanges with a third conformation as well. In addition to the kinetics of the exchange reaction, methyl carbon chemical shifts of the excited state are also obtained from analysis of the (13)C CEST data.