Allosteric Coupling of Drug Binding and Intracellular Signaling in the A2A Adenosine Receptor.

Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A2A adenosine receptor (A2AAR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15N-1H NMR signals in A2AAR were individually assigned. These NMR probes provided insight into the role of Asp522.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 2466.48, and delineated the structural response to variable efficacy of bound drugs across A2AAR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp522.50.

Hsp90-Tau complex reveals molecular basis for specificity in chaperone action.

Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is poorly understood. Here, we obtained a structural model of Hsp90 in complex with its natural disease-associated substrate, the intrinsically disordered Tau protein. Hsp90 binds to a broad region in Tau that includes the aggregation-prone repeats. Complementarily, a 106-Å-long substrate-binding interface in Hsp90 enables many low-affinity contacts. This allows recognition of scattered hydrophobic residues in late folding intermediates that remain after early burial of the Hsp70 sites. Our model resolves the paradox of how Hsp90 specifically selects for late folding intermediates but also for some intrinsically disordered proteins-through the eyes of Hsp90 they look the same.

A2A adenosine receptor functional states characterized by 19F-NMR.

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A2A adenosine receptor (A2AAR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used 19F-NMR experiments with probes at the A2AAR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A2AAR and one inactive substate that is not seen in complexes with agonists, suggesting that A2AAR activation includes both induced fit and conformational selection mechanisms. Comparison of A2AAR and a constitutively active mutant established relations between the 19F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant.

NMR structure and dynamics of the agonist dynorphin peptide bound to the human kappa opioid receptor.

The structure of the dynorphin (1-13) peptide (dynorphin) bound to the human kappa opioid receptor (KOR) has been determined by liquid-state NMR spectroscopy. (1)H and (15)N chemical shift variations indicated that free and bound peptide is in fast exchange in solutions containing 1 mM dynorphin and 0.01 mM KOR. Radioligand binding indicated an intermediate-affinity interaction, with a Kd of ∼200 nM. Transferred nuclear Overhauser enhancement spectroscopy was used to determine the structure of bound dynorphin. The N-terminal opioid signature, YGGF, was observed to be flexibly disordered, the central part of the peptide from L5 to R9 to form a helical turn, and the C-terminal segment from P10 to K13 to be flexibly disordered in this intermediate-affinity bound state. Combining molecular modeling with NMR provided an initial framework for understanding multistep activation of a G protein-coupled receptor by its cognate peptide ligand.

Non-Uniform Sampling and J-UNIO Automation for Efficient Protein NMR Structure Determination.

High-resolution structure determination of small proteins in solution is one of the big assets of NMR spectroscopy in structural biology. Improvements in the efficiency of NMR structure determination by advances in NMR experiments and automation of data handling therefore attracts continued interest. Here, non-uniform sampling (NUS) of 3D heteronuclear-resolved [(1)H,(1)H]-NOESY data yielded two- to three-fold savings of instrument time for structure determinations of soluble proteins. With the 152-residue protein NP_372339.1 from Staphylococcus aureus and the 71-residue protein NP_346341.1 from Streptococcus pneumonia we show that high-quality structures can be obtained with NUS NMR data, which are equally well amenable to robust automated analysis as the corresponding uniformly sampled data.

Hsp90 structure and function studied by NMR spectroscopy.

The molecular chaperone Hsp90 plays a crucial role in folding and maturation of regulatory proteins. Key aspects of Hsp90's molecular mechanism and its adenosine-5'-triphosphate (ATP)-controlled active cycle remain elusive. In particular the role of conformational changes during the ATPase cycle and the molecular basis of the interactions with substrate proteins are poorly understood. The dynamic nature of the Hsp90 machine designates nuclear magnetic resonance (NMR) spectroscopy as an attractive method to unravel both the chaperoning mechanism and interaction with partner proteins. NMR is particularly suitable to provide a dynamic picture of protein-protein interactions at atomic resolution. Hsp90 is rather a challenging protein for NMR studies, due to its high molecular weight and its structural flexibility. The recent technologic advances allowed overcoming many of the traditional obstacles. Here, we describe the different approaches that allowed the investigation of Hsp90 using state-of-the-art NMR methods and the results that were obtained. NMR spectroscopy contributed to understanding Hsp90's interaction with the co-chaperones p23, Aha1 and Cdc37. A particular exciting prospect of NMR, however, is the analysis of Hsp90 interaction with substrate proteins. Here, the ability of this method to contribute to the structural characterization of not fully folded proteins becomes crucial. Especially the interaction of Hsp90 with one of its natural clients, the tumour suppressor p53, has been intensively studied by NMR spectroscopy. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).

ß2-Adrenergic Receptor Conformational Response to Fusion Protein in the Third Intracellular Loop.

Fluorine-19 nuclear magnetic resonance (NMR) was used to study conformational equilibria at the intracellular tips of helices VI and VII in a variant ß2-adrenergic receptor (ß2AR) containing T4-lysozyme fused into the third intracellular loop (ß2AR-T4L), a G protein-coupled receptor (GPCR) modification widely used in crystal structure determination. G-protein signaling at helix VI showed nearly complete population of an active-like state for all ligand efficacies in the absence of an intracellular protein. For arrestin signaling at helix VII, a native-like equilibrium was observed, except for complexes with ligands devoid of a hydrophobic moiety at the ethanolamine end. These data confirm that response of G-protein and arrestin signaling to ligand efficacy is not coupled, and presents evidence for long-range effects between fusion protein and orthosteric binding cavity, which are suppressed by voluminous bound ligands. Solution NMR thus provides complementary information, which should be considered in functional interpretations of GPCR crystal structures obtained with ICL3 fusions.

Fluorine-19 NMR of integral membrane proteins illustrated with studies of GPCRs.

Fluorine-19 is a spin-½ NMR isotope with high sensitivity and large chemical shift dispersion, which makes it attractive for high resolution NMR spectroscopy in solution. For studies of membrane proteins it is further of interest that (19)F is rarely found in biological materials, which enables observation of extrinsic (19)F labels with minimal interference from background signals. Today, after a period with rather limited use of (19)F NMR in structural biology, we witness renewed interest in this technology for studies of complex supramolecular systems. Here we report on recent (19)F NMR studies with the G protein-coupled receptor family of membrane proteins.

3D DOSY-TROSY to determine the translational diffusion coefficient of large protein complexes.

The translational diffusion coefficient is a sensitive parameter to probe conformational changes in proteins and protein-protein interactions. Pulsed-field gradient NMR spectroscopy allows one to measure the translational diffusion with high accuracy. Two-dimensional (2D) heteronuclear NMR spectroscopy combined with diffusion-ordered spectroscopy (DOSY) provides improved resolution and therefore selectivity when compared with a conventional 1D readout. Here, we show that a combination of selective isotope labelling, 2D ¹H-¹³C methyl-TROSY (transverse relaxation-optimised spectroscopy) and DOSY allows one to study diffusion properties of large protein complexes. We propose that a 3D DOSY-heteronuclear multiple quantum coherence (HMQC) pulse sequence, that uses the TROSY effect of the HMQC sequence for ¹³C methyl-labelled proteins, is highly suitable for measuring the diffusion coefficient of large proteins. We used the 20 kDa co-chaperone p23 as model system to test this 3D DOSY-TROSY technique under various conditions. We determined the diffusion coefficient of p23 in viscous solutions, mimicking large complexes of up to 200 kDa. We found the experimental data to be in excellent agreement with theoretical predictions. To demonstrate the use for complex formation, we applied this technique to record the formation of a complex of p23 with the molecular chaperone Hsp90, which is around 200 kDa. We anticipate that 3D DOSY-TROSY will be a useful tool to study conformational changes in large protein complexes.

Critical scaffolding regions of the tumor suppressor Axin1 are natively unfolded.

The Wnt pathway tumor-suppressor protein Axin coordinates the formation of a critical multiprotein destruction complex that serves to downregulate ß-catenin protein levels, thereby preventing target gene activation. Given the lack of structural information on some of the major functional parts of Axin, it remains unresolved how the recruitment and positioning of Wnt pathway kinases, such as glycogen synthase kinase 3ß, are coordinated to bring about ß-catenin phosphorylation. Using various biochemical and biophysical methods, we demonstrate here that the central region of Axin that is implicated in binding glycogen synthase kinase 3ß and ß-catenin is natively unfolded. Our results support a model in which the unfolded nature of these critical scaffolding regions in Axin facilitates dynamic interactions with a kinase and its substrate, which in turn act upon each other.