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.

DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular milieu.

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition. We found that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells.

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs of in-cell experiments. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP-assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

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.

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.

Visualizing the molecular recognition trajectory of an intrinsically disordered protein using multinuclear relaxation dispersion NMR.

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX. This helix then locates into the binding site at a rate coincident with intrinsic breathing motions of the helical groove on the surface of PX. The binding kinetics of complex formation are thus regulated by the intrinsic free-state conformational dynamics of both proteins. This approach, providing high-resolution structural and kinetic information about a complex folding and binding interaction trajectory, can be applied to a number of experimental systems to provide a general framework for understanding conformational disorder in biomolecular function.

Ensemble Calculation for Intrinsically Disordered Proteins Using NMR Parameters.

Intrinsically disordered proteins (IDPs) perform their function despite their lack of well-defined tertiary structure. Residual structure has been observed in IDPs, commonly described as transient/dynamic or expressed in terms of fractional populations. In order to understand how the protein primary sequence dictates the dynamic and structural properties of IDPs and in general to understand how IDPs function, atomic-level descriptions are needed. Nuclear magnetic resonance spectroscopy provides information about local and long-range structure in IDPs at amino acid specific resolution and can be used in combination with ensemble descriptions to represent the dynamic nature of IDPs. In this chapter we describe sample-and-select approaches for ensemble modelling of local structural propensities in IDPs with specific emphasis on validation of these ensembles.

Conformational propensities of intrinsically disordered proteins from NMR chemical shifts.

The realization that a protein can be fully functional even in the absence of a stable three-dimensional structure has motivated a large number of studies describing the conformational behaviour of these proteins at atomic resolution. Here, we review recent advances in the determination of local structural propensities of intrinsically disordered proteins (IDPs) from experimental NMR chemical shifts. A mapping of the local structure in IDPs is of paramount importance in order to understand the molecular details of complex formation, in particular, for IDPs that fold upon binding or undergo structural transitions to pathological forms of the same protein. We discuss experimental strategies for the spectral assignment of IDPs, chemical shift prediction algorithms and the generation of representative structural ensembles of IDPs on the basis of chemical shifts. Additionally, we highlight the inherent degeneracies associated with the determination of IDP sub-state populations from NMR chemical shifts alone.

Conformational ensembles explain NMR spectra of frozen intrinsically disordered proteins.

Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble. We validated our simulations by comparing them to spectra of α-synuclein acquired under different experimental conditions. Our assignments of peaks to discrete dihedral angle populations suggest that structural constraints are attainable under cryogenic conditions. The ability to infer ensemble populations from peak shapes has important implications for DNP MAS NMR studies of proteins with regions of disorder in living cells because chemical shifts are the most accessible measured parameter.

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells.

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble. Moreover, because the DNP polarization agent can be dispersed both homogenously and inhomogenously throughout the cellular biomass, in cell DNP-assisted solid state NMR experiments can report either quantitatively upon the structural ensemble or can preferentially report upon the structural ensemble with a spatial bias. Using DNP-assisted MAS NMR we establish that the spectra of purified α-syn in the membrane-associated and intrinsically disordered forms have distinguishable spectra. When the polarization agent is introduced into cells by electroporation and dispersed homogenously, a minority of the α-syn inside HEK293 cells adopts a highly α-helical rich conformation. Alteration of the spatial distribution of the polarization agent preferentially enhances the signal from molecules nearer to the cellular periphery, thus the α-helical rich population is preferentially adopted toward the cellular periphery. This demonstrates how selectively altering the spatial distribution of the DNP polarization agent can be a powerful tool for preferential reporting on specific structural ensembles, paving the way for more nuanced investigations into the conformations that proteins adopt in different areas of the cell.

Enthalpy-Entropy Compensation in the Promiscuous Interaction of an Intrinsically Disordered Protein with Homologous Protein Partners.

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.

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability.

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Small-angle X-ray scattering- and nuclear magnetic resonance-derived conformational ensemble of the highly flexible antitoxin PaaA2.

Antitoxins from prokaryotic type II toxin-antitoxin modules are characterized by a high degree of intrinsic disorder. The description of such highly flexible proteins is challenging because they cannot be represented by a single structure. Here, we present a combination of SAXS and NMR data to describe the conformational ensemble of the PaaA2 antitoxin from the human pathogen E. coli O157. The method encompasses the use of SAXS data to filter ensembles out of a pool of conformers generated by a custom NMR structure calculation protocol and the subsequent refinement by a block jackknife procedure. The final ensemble obtained through the method is validated by an established residual dipolar coupling analysis. We show that the conformational ensemble of PaaA2 is highly compact and that the protein exists in solution as two preformed helices, connected by a flexible linker, that probably act as molecular recognition elements for toxin inhibition.