Highlights d NMR spectroscopy and mutagenesis analysis provide insight on ghrelin binding mode d Residues outside of the known binding motif are identified d Ghrelin binds via an extended surface to its G proteincoupled receptor d Hybrid method of modeling peptide-binding receptors is described

Background: Disulfide-regulated NTPDases from T. gondii are related to the virulence of the parasite. Results: Crystal structures of the active and inactive state were determined. Conclusion: The 258 -268 disulfide bridge acts like a clamp, which upon reduction allows concerted motions of the subunits and domains of the tetrameric enzyme. Significance: First molecular model of the disulfide-regulated activation mode.

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.

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 13C‐labeled. NMR‐signal assignment was achieved by dynamic‐nuclear‐polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13C 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.

Summary 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 which represent the experimental distance distributions best 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).