Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements.

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron-electron resonance (DEER) pulsed EPR technique on the receptor.

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13 C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13 C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp11623.50 was identified.

The dynamics of the G protein-coupled neuropeptide Y2 receptor in monounsaturated membranes investigated by solid-state NMR spectroscopy.

In contrast to the static snapshots provided by protein crystallography, G protein-coupled receptors constitute a group of proteins with highly dynamic properties, which are required in the receptors' function as signaling molecule. Here, the human neuropeptide Y2 receptor was reconstituted into a model membrane composed of monounsaturated phospholipids and solid-state NMR was used to characterize its dynamics. Qualitative static (15)N NMR spectra and quantitative determination of (1)H-(13)C order parameters through measurement of the (1)H-(13)C dipolar couplings of the CH, CH2 and CH3 groups revealed axially symmetric motions of the whole molecule in the membrane and molecular fluctuations of varying amplitude from all molecular segments. The molecular order parameters (S(backbone) = 0.59-0.67, S(CH2) = 0.41-0.51 and S(CH3) = 0.22) obtained in directly polarized (13)C NMR experiments demonstrate that the Y2 receptor is highly mobile in the native-like membrane. Interestingly, according to these results the receptor was found to be slightly more rigid in the membranes formed by the monounsaturated phospholipids than by saturated phospholipids as investigated previously. This could be caused by an increased chain length of the monounsaturated lipids, which may result in a higher helical content of the receptor. Furthermore, the incorporation of cholesterol, phosphatidylethanolamine, or negatively charged phosphatidylserine into the membrane did not have a significant influence on the molecular mobility of the Y2 receptor.

Structural insight into activation mechanism of Toxoplasma gondii nucleoside triphosphate diphosphohydrolases by disulfide reduction.

The intracellular parasite Toxoplasma gondii produces two nucleoside triphosphate diphosphohydrolases (NTPDase1 and -3). These tetrameric, cysteine-rich enzymes require activation by reductive cleavage of a hitherto unknown disulfide bond. Despite a 97% sequence identity, both isozymes differ largely in their ability to hydrolyze ATP and ADP. Here, we present crystal structures of inactive NTPDase3 as an apo form and in complex with the product AMP to resolutions of 2.0 and 2.2 Å, respectively. We find that the enzyme is present in an open conformation that precludes productive substrate binding and catalysis. The cysteine bridge 258-268 is identified to be responsible for locking of activity. Crystal structures of constitutively active variants of NTPDase1 and -3 generated by mutation of Cys(258)-Cys(268) show that opening of the regulatory cysteine bridge induces a pronounced contraction of the whole tetramer. This is accompanied by a 12° domain closure motion resulting in the correct arrangement of all active site residues. A complex structure of activated NTPDase3 with a non-hydrolyzable ATP analog and the cofactor Mg(2+) to a resolution of 2.85 Å indicates that catalytic differences between the NTPDases are primarily dictated by differences in positioning of the adenine base caused by substitution of Arg(492) and Glu(493) in NTPDase1 by glycines in NTPDase3.

The crystal structure of Toxoplasma gondii nucleoside triphosphate diphosphohydrolase 1 represents a conformational intermediate in the reductive activation mechanism of the tetrameric enzyme.

Toxoplasma gondii nucleoside triphosphate diphosphohydrolase (NTPDase) 1 was crystallized in an intermediate tetrameric conformation. The crystal structure is similar to that of T. gondii NTPDase3 and represents an inactive conformation as the activating disulfide bridge is not reduced and the active site cleft between the two domains of each monomer is open. However, the arrangement of the monomers within the tetramer differs from that of the inactive form of NTPDase3 and may represent an intermediate conformation on the path of the closure motion of the tetramer induced upon activation.

The ATP/ADP substrate specificity switch between Toxoplasma gondii NTPDase1 and NTPDase3 is caused by an altered mode of binding of the substrate base.

Two nucleoside triphosphate diphosphohydrolase isoforms (NTPDase1 and NTPDase3) are responsible for the hydrolysis of nucleotides by the intracellular protozoan Toxoplasma gondii. They constitute about 3 % of the total parasite protein. Despite sharing 97 % sequence identity they exhibit opposite ATP versus ADP substrate discrimination ratios. Here we show by mutagenesis that the residues G492/G493 in NTPDase3 and R492/E493 in NTPDase1 are predominantly responsible for the differences in substrate specificity. Crystal structures of NTPDase1 in complexation with analogues of ATP and ADP reveal that the inverted substrate specificity of NTPDase1 relative to NTPDase3 is achieved by switching from the canonical substrate binding mode to a very different alternative one. Instead of being stacked on top of a helix of the C-terminal domain the nucleotide base is positioned in the interdomain space between the side chains of R108 and R492, recruited from both domains. Furthermore, we show that the NTPDase1 substrate specificity is mainly dependent on the presence of the side chain of E493, which causes repositioning of the ribose component of the nucleotide. All in all, binding by the flexible side chains in the alternative binding mode in NTPDase1 allows for equally good positioning of ATP and ADP with increased activity toward ADP relative to what is seen in the case of NTPDase3.

Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor.

Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for 13C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane α-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged 1H-13C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly 13C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions.

Structures of 2-Hydroxyisobutyric Acid-CoA Ligase Reveal Determinants of Substrate Specificity and Describe a Multi-Conformational Catalytic Cycle.

2-Hydroxyisobutyric acid (2-HIBA) is a biomarker of adiposity and associated metabolic diseases such as diabetes mellitus. It is also formed in the bacterial degradation pathway of the fuel oxygenate methyl tert-butyl ether (MTBE), requiring thioesterification with CoA prior to isomerization to 3-hydroxybutyryl-CoA by B12-dependent acyl-CoA mutases. Here, we identify the adenylating enzymes superfamily member 2-HIBA-CoA ligase (HCL) in the MTBE-degrading bacterium Aquincola tertiaricarbonis L108 by knockout experiments. To characterize this central enzyme of 2-HIBA metabolism, ligase activity kinetics of purified HCL and its X-ray crystal structures were studied. We analyzed the enzyme in three states, which differ in the orientation of the two enzyme domains. A 154° rotation of the C-terminal domain accompanies the switch from the adenylate- into the thioester-forming state. Furthermore, a third conformation was obtained, which differs by 50° and 130° from the adenylation and thioesterification states, respectively. Phylogenetic and structural analysis reveals that HCL defines a new subgroup within phenylacetate-CoA ligases (PCLs) thus far described to exclusively accept aromatic acyl substrates. In contrast, kinetic characterization clearly demonstrated that HCL catalyzes CoA activation of several aliphatic short-chain carboxylic acids, preferentially 2-HIBA. Compared to the classical PCL representatives PaaK1 and PaaK2 of Burkholderia cenocepacia J2315, the acyl binding pocket of HCL is significantly smaller and more polar, due to unique active-site residues Y164 and S239 forming H-bonds with the OH-group of the acyl substrate moiety. Furthermore, HCL and PaaK topologies illustrate the evolutionary steps leading from a homodimeric to the fused monomeric core fold found in other ligases.

Structural Model of Ghrelin Bound to its G Protein-Coupled Receptor.

The peptide ghrelin targets the growth hormone secretagogue receptor 1a (GHSR) to signal changes in cell metabolism and is a sought-after therapeutic target, although no structure is known to date. To investigate the structural basis of ghrelin binding to GHSR, we used solid-state nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, and Rosetta modeling. The use of saturation transfer difference NMR identified key residues in the peptide for receptor binding beyond the known motif. This information combined with assignment of the secondary structure of ghrelin in its receptor-bound state was incorporated into Rosetta using an approach that accounts for flexible binding partners. The NMR data and models revealed an extended binding surface that was confirmed via mutagenesis. Our results agree with a growing evidence of peptides interacting via two sites at G protein-coupled receptors.

Characterization of the Domain Orientations of E. coli 5'-Nucleotidase by Fitting an Ensemble of Conformers to DEER Distance Distributions.

Escherichia coli 5'-nucleotidase is a two-domain enzyme exhibiting a unique 96° domain motion that is required for catalysis. Here we present an integrated structural biology study that combines DEER distance distributions with structural information from X-ray crystallography and computational biology to describe the population of presumably almost isoenergetic open and closed states in solution. Ensembles of models that best represent the experimental distance distributions are determined by a Monte Carlo search algorithm. As a result, predominantly open conformations are observed in the unliganded state indicating that the majority of enzyme molecules await substrate binding for the catalytic cycle. The addition of a substrate analog yields ensembles with an almost equal mixture of open and closed states. Thus, in the presence of substrate, efficient catalysis is provided by the simultaneous appearance of open conformers (binding substrate or releasing product) and closed conformers (enabling the turnover of the substrate).

Contribution of the two domains of E. coli 5'-nucleotidase to substrate specificity and catalysis.

Escherichia coli 5'-nucleotidase, a two-domain enzyme, dephosphorylates various nucleotides with comparable efficiency. We have expressed the two domains individually in E. coli and show by liquid state NMR that they are properly folded. Kinetic characterization reveals that the C-terminal domain, which contains the substrate-binding pocket, is completely inactive while the N-terminal domain with the two-metal-ion-center and the core catalytic residues exhibits significant activity, especially towards substrates with activated phosphate bonds (ATP, ADP, p-nitrophenyl phosphate). In contrast, residues of the C-terminal domain are required for efficient hydrolysis of AMP.