Reto Horst

Biased signaling pathways in β2-adrenergic receptor characterized by 19f-nmr

Choosing a Path The β2-adrenergic receptor (β2AR) is a G protein–coupled receptor that recognizes diverse ligands to trigger signaling in the cell. Besides binding G proteins, activated β2AR can be phosphorylated and bind arrestin, which redirects signaling to other pathways. Some β2AR ligands are “biased” in that they differentially activate G protein or arrestin signaling. Liu et al. (p. 1106, published online 19 January; see the Perspective by Sprang and Chief Elk) used 19F-NMR spectroscopy to examine conformational changes associated with a range of ligands and discovered that biased ligands caused differential shifts in equilibrium between two conformational states—the G protein binding state and the arrestin binding state—and thus provide a basis for rational design of pharmacological ligands. Selective effects of different ligands provide insights into the structural plasticity of receptor signaling. Extracellular ligand binding to G protein–coupled receptors (GPCRs) modulates G protein and β-arrestin signaling by changing the conformational states of the cytoplasmic region of the receptor. Using site-specific 19F-NMR (fluorine-19 nuclear magnetic resonance) labels in the β2-adrenergic receptor (β2AR) in complexes with various ligands, we observed that the cytoplasmic ends of helices VI and VII adopt two major conformational states. Changes in the NMR signals reveal that agonist binding primarily shifts the equilibrium toward the G protein–specific active state of helix VI. In contrast, β-arrestin–biased ligands predominantly impact the conformational states of helix VII. The selective effects of different ligands on the conformational equilibria involving helices VI and VII provide insights into the long-range structural plasticity of β2AR in partial and biased agonist signaling.clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): May 17, 2012 www.sciencemag.org (this information is current as of The following resources related to this article are available online at

NMR structure reveals intramolecular regulation mechanism for pheromone binding and release

Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBPA present at pH 4.5 and BmPBPB present at pH 6.5. Here, we describe the NMR structure of BmPBPA, which consists of a tightly packed arrangement of seven α-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four α-helices that forms the ligand binding site in the crystal structure of a BmPBP–pheromone complex is preserved in BmPBPA. The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, α7, which is located in the pheromone-binding site in the core of the unliganded BmPBPA. Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of α7 triggering the release of the pheromone from BmPBP to the membrane-standing receptor.

Structural basis of chaperone-subunit complex recognition by the type 1 pilus assembly platform FimD

Adhesive type 1 pili from uropathogenic Escherichia coli are filamentous protein complexes that are attached to the assembly platform FimD in the outer membrane. During pilus assembly, FimD binds complexes between the chaperone FimC and type 1 pilus subunits in the periplasm and mediates subunit translocation to the cell surface. Here we report nuclear magnetic resonance and X‐ray protein structures of the N‐terminal substrate recognition domain of FimD (FimDN) before and after binding of a chaperone–subunit complex. FimDN consists of a flexible N‐terminal segment of 24 residues, a structured core with a novel fold, and a C‐terminal hinge segment. In the ternary complex, residues 1–24 of FimDN specifically interact with both FimC and the subunit, acting as a sensor for loaded FimC molecules. Together with in vivo complementation studies, we show how this mechanism enables recognition and discrimination of different chaperone–subunit complexes by bacterial pilus assembly platforms.

NMR structure of the unliganded Bombyx mori pheromone-binding protein at physiological pH

The nuclear magnetic resonance structure of the unliganded pheromone‐binding protein (PBP) from Bombyx mori at pH above 6.5, BmPBPB, consists of seven helices with residues 3–8, 16–22, 29–32, 46–59, 70–79, 84–100, and 107–124, and contains the three disulfide bridges 19–54, 50–108, and 97–117. This polypeptide fold encloses a large hydrophobic cavity, with a sufficient volume to accommodate the natural ligand bombykol. The polypeptide folds in free BmPBPB and in crystals of a BmPBP–bombykol complex are nearly identical, indicating that the B‐form of BmPBP in solution represents the active conformation for ligand binding.

Folding trajectories of human dihydrofolate reductase inside the GroEL–GroES chaperonin cavity and free in solution

The chaperonin GroEL binds non-native polypeptides in an open ring via hydrophobic contacts and then, after ATP and GroES binding to the same ring as polypeptide, mediates productive folding in the now hydrophilic, encapsulated cis chamber. The nature of the folding reaction in the cis cavity remains poorly understood. In particular, it is unclear whether polypeptides take the same route to the native state in this cavity as they do when folding spontaneously free in solution. Here, we have addressed this question by using NMR measurements of the time course of acquisition of amide proton exchange protection of human dihydrofolate reductase (DHFR) during folding in the presence of methotrexate and ATP either free in solution or inside the stable cavity formed between a single ring variant of GroEL, SR1, and GroES. Recovery of DHFR refolded by the SR1/GroES-mediated reaction is 2-fold higher than in the spontaneous reaction. Nevertheless, DHFR folding was found to proceed by the same trajectories inside the cis folding chamber and free in solution. These observations are consistent with the description of the chaperonin chamber as an “Anfinsen cage” where polypeptide folding is determined solely by the amino acid sequence, as it is in solution. However, if misfolding occurs in the confinement of the chaperonin cavity, the polypeptide chain cannot undergo aggregation but rather finds its way back to a productive pathway in a manner that cannot be accomplished in solution, resulting in the observed high overall recovery.

Microscale NMR Screening of New Detergents for Membrane Protein Structural Biology

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly (2)H, (15)N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ (15)N, (1)H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.

Nuclear Magnetic Resonance Structure of the N-Terminal Domain of Nonstructural Protein 3 from the Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four β-strands and two α-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

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.

β₂-adrenergic receptor activation by agonists studied with ¹⁹F NMR spectroscopy.

G-protein-coupled receptors (GPCRs) recognize a wide array of orthosteric ligands in their binding site on the periplasmic cell membrane surface, initiating signal transmission through the cellular membrane to cytoplasmic partner proteins. Crystal structures of several human GPCRs in complexes with antagonists and agonists provide insights into activation-related structural rearrangements,[1] and fluorescence spectroscopy experiments indicated activation-related conformational changes in detergent-solubilized receptors.[2] 19F-NMR spectroscopy and site-specific mutagenesis, as applied previously with mammalian rhodopsin,[3] more recently revealed an equilibrium between an activated state A and an inactive state I in the β2-adrenergic receptor (β2AR).[4] This communication now presents thermodynamic and kinetic data for this conformational equilibrium in β2AR. In our earlier experiments,[4] the β2AR complexes were reconstituted in mixed micelles of n-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS), with DDM:CHS = 5:1, and 19F-labels were introduced by conjugation of 2,2,2-trifluoroethanethiol (TET) with cysteines near the cytoplasmic ends of the helices VI (Cys265) and VII (Cys327), and at the C-terminus (Cys341). Ligand binding assays showed that the labeled proteins retained the biological activity.[4] Sequence-specific 19F-NMR assignments were based on comparison of β2AR variants with single-residue TET-labeling, and the signal I was assigned from its high intensity in the apo-form of β2AR and its complexes with inverse agonists.[4] Observation of the TET labels in β2AR-complexes with different pharmacological ligands then enabled to distinguish between the activation of two different signaling pathways.[4] A first extension of the previous work was to analyze the temperature dependence of the 1D 19F-NMR spectra of β2AR (TETC265, C327S, C341A) and β2AR (C265A, TETC327, C341A) in terms of the thermodynamic parameters that characterize the conformational equilibrium between the states A and I. This analysis was focused on complexes with agonists, i.e., norepinephrine and formoterol, since for the complexes with antagonists or inverse agonists the amplitude of the signal A is near the noise level and its volume cannot reliably be quantified. Based on the observation that the temperature dependence over the range 280 K to 310 K of the NMR spectra recorded with the agonist complexes was reversible, the relative populations of the conformations represented by the signals A and I, pA and pI, were determined by fits to a double-Lorentzian function (Figure 1), yielding an apparent equilibrium constant, K = pI/ pA. ln K was found to depend linearly on the inverse of the temperature, T (Figure 2), which is in agreement with the van’t Hoff relationship between K, the molar enthalpy difference, ΔH0, and the molar entropy difference, ΔS0.[5] lnK=−(ΔH0−TS0)/RT, (1) where R is the gas constant. ΔH0 values near 40 kJ/mol were obtained for both labeling sites at Cys265 and Cys327, and for both agonists used (Table 1), suggesting that the structural differences between the states A and I observed in the 19F-NMR spectra (Figure 1) represent more extensive conformational rearrangements than, for example, reorientation of a single amino acid side chain. For the different systems in Table 1 the Gibbs free energy, ΔG0 = −ln(K)RT, is between 0 and 3 kJ/mol, which shows that the entropy and enthalpy terms (Table 1) nearly cancel each other. This observation is in line with the widely observed entropy–enthalpy compensation in biological systems.[6] Figure 1 1D 19F-NMR spectra at 280 K, 298 K and 310 K of the complexes with the partial agonist norepinephrine (upper row) and the full agonist formoterol (lower row) of β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) in mixed micelles of ... Figure 2 van’t Hoff plots for the interconversion between the active state (A) and the inactive state (I) of β2AR(TETC265, C327S, C341A) and β2AR(C265A, TETC327, C341A) in the complexes with norepinephrine and formoterol. The apparent equilibrium constants, ... Table 1 Molar enthalpy differences, ΔH0, and molar entropy differences, ΔS0, for the interconversion between the activated and inactive states of the formoterol and norepinephrine complexes of β2AR (TETC265, C327S, C341A) and β ... To investigate the exchange rates between the states A and I within the framework of a 2-state model, where k1 and k−1 are the forward and reverse rate constants, A⇄k−1k1I, (2) we used 2D 19F–19F exchange spectroscopy (EXSY)[7] and 1D 19F saturation transfer NMR experiments.[8] The overall exchange rate constant, kex, is given by kex=k1+k−1=k1/pI=k−1/pA, (3) where pA and pI are the relative populations of the states A and I. The observation of two distinct signals A and I in the 19F-NMR spectra of β2AR (Figure 1) showed that the conformational exchange is slow on the 19F-NMR chemical shift time scale, so that kex satisfies the inequality, kex≪Δω2pApI, (4) where Δω is the chemical shift between I and A in rad/sec.[9] For TET-labeled Cys265 and Cys327 the Δω values are 2×103 rad/sec and 4×103 rad/sec, respectively, and pA = 1−pI is between 0.2 and 0.9.[4] An upper limit of kex ≤ 103 s−1 was thus previously established, and additional support for this limit was obtained from experiments with paramagnetic shift reagents.[4] Here, 2D [19F,19F]-EXSY experiments with a TET β2AR–isoproterenol complex were performed with mixing times of 300 and 600 ms. For kex values of 10 s−1 or larger, 2D [19F,19F]-cross-peaks between the signals A and I are predicted to be of similar size as the diagonal peaks in these experiments (Figure 3c). The absence of [19F,19F]-cross-peaks (Figure 3) then enabled us to establish a new upper limit of kex < 10 s−1 at 280 K. 19F-NMR saturation-transfer experiments with apo- β2AR(C265A, TETC327, C341A) at 280 K further indicated that the exchange rate is significantly slower than 10 s−1. Considering the spectral overlap of the two signals (Figure 4), we applied selective off-resonance continuous wave (cw) pre-irradiation in these experiments (Figure 4), and analyzed the resulting intensity variations of the signals A and I with model simulations based on the Bloch equations for two-site exchange (Equations (5) to (10) in the Appendix).[10] The longitudinal and transverse spin relaxation times, T1 and T2, used in these model computations were determined with an inversion–recovery experiment (see the Experimental Section), and from the line shapes of the signals in the 1D 19F-NMR spectrum (top trace in Figure 4), respectively. Comparison of the experimental data with the simulations (Figure 5) showed that the observed decay of the signal I was due to direct saturation by the off-resonance irradiation, and that there was no measureable contribution due to coherence transfer from signal A to signal I by conformational exchange (Figure 5). Figure 3 2D [19F,19F]-EXSY experiments with the isoproterenol complex of wild type β2AR (TETC265, TETC327, TETC341) in mixed micelles of DDM and CHS 5:1. (a) Contour plot. At the top the chemical shifts of five previously assigned peaks[4] are indicated, ... Figure 4 1D 19F-NMR saturation transfer experiments with apo-β2AR(C265A, TETC327, C341A) in mixed micelles of DDM and CHS 5:1 used to measure the exchange rate between activated state (A) and inactive state (I) of β2AR. Top trace: 1D 19F-NMR spectrum, ... Figure 5 Model simulations of the attenuation of the intensity of signal I in the NMR spectra of Figure 4 by off-resonance continuous wave (cw) pre-irradiation at the chemical shifts i to v. The relative peak volumes of the signal I in 1D 19F-NMR saturation transfer experiments, ... In conclusion, this paper used TET 19F-NMR probes attached to three cysteine residues near the cytoplasmic surface to determine thermodynamic and kinetic parameters for the equilibrium between an active state, A, and an inactive state, I, of β2AR, which both represent an ensemble of rapidly interconverting conformers. Slow exchange, on the TET 19F-NMR chemical shift timescale, between the states I and A enabled a quantitative characterization of this rate process. Large values for ΔH0 (Table 1) and an exchange rate slower than 10 S−1 (Figures 3–5) indicate that the interconversion entails major structural rearrangements, which likely involve polypeptide backbone segments.[11] Furthermore, the near-identical values of ΔH0 for different ligands bound to the receptor (Table 1) indicate that the equilibrium between the two states is an intrinsic property of the receptor, so that binding of different orthosteric ligands, allosteric effectors, and possibly of cytoplasmic partner proteins would result in shifts of this pre-existing equilibrium. Comparison with recent related studies of β2AR in DDM micelles shows that TET-labeling provides different information from NMR experiments using either a different 19F-label, 3-bromo-1,1,1-trifluoroacetone (BTFA), on Cys 265[12] or 13C-labeled Met 82,[13] which both provided evidence for two or multiple states of β2AR in fast exchange on the respective chemical shift time scales. Results obtained by combining BTFA-labeling of Cys 265 with the use of the detergent maltose-neopentyl-glycol (MNG-3) were interpreted in terms of slow exchange between at least three states of β2AR.[12] Different experimental approaches thus appear to provide complementary information on the β2AR system, and one can look forward to continued studies of the dynamics of GPCRs with a variety of different reporter groups, including investigations of possible modulation of the protein conformational equilibria by allosteric effectors.

Proton–proton Overhauser NMR spectroscopy with polypeptide chains in large structures

The use of 1H–1H nuclear Overhauser effects (NOE) for structural studies of uniformly deuterated polypeptide chains in large structures is investigated by model calculations and NMR experiments. Detailed analysis of the evolution of the magnetization during 1H–1H NOE experiments under slow-motion conditions shows that the maximal 1H–1H NOE transfer is independent of the overall rotational correlation time, even in the presence of chemical exchange with the bulk water, provided that the mixing time is adjusted for the size of the structure studied. 1H–1H NOE buildup measurements were performed for the 472-kDa complex of the 72-kDa cochaperonin GroES with a 400-kDa single-ring variant of the chaperonin GroEL (SR1). These experiments demonstrate that multidimensional NOESY experiments with cross-correlated relaxation-enhanced polarization transfer and transverse relaxation-optimized spectroscopy elements can be applied to structures of molecular masses up to several hundred kilodaltabs, which opens new possibilities for studying functional interactions in large maromolecular assemblies in solution.