Background: It remains unclear why vasopressin induces greater antidiuresis through V2R than does oxytocin. Results: Vasopressin sustains cAMP signaling during V2R internalization, a process promoted by ␤-arrestins, and is halted by the retromer complex. Conclusion: This new noncanonical model of GPCR signaling differentiates the actions of vasopressin and oxytocin.Significance: This emerging model may explain the physiological bias between ligands.

G protein-coupled receptors (GPCRs) participate in ubiquitous transmembrane signal transduction processes by activating heterotrimeric G proteins. In the current "canonical" model of GPCR signaling, arrestins terminate receptor signaling by impairing receptor-G-protein coupling and promoting receptor internalization. However, parathyroid hormone receptor type 1 (PTHR), an essential GPCR involved in bone and mineral metabolism, does not follow this conventional desensitization paradigm. β-Arrestins prolong G protein (G S )-mediated cAMP generation triggered by PTH, a process that correlates with the persistence of arrestin-PTHR complexes on endosomes and which is thought to be associated with prolonged physiological calcemic and phosphate responses. This presents an inescapable paradox for the current model of arrestin-mediated receptor-G-protein decoupling. Here we show that PTHR forms a ternary complex that includes arrestin and the Gβγ dimer in response to PTH stimulation, which in turn causes an accelerated rate of G S activation and increases the steady-state levels of activated G S , leading to prolonged generation of cAMP. This work provides the mechanistic basis for an alternative model of GPCR signaling in which arrestins contribute to sustaining the effect of an agonist hormone on the receptor.

The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiationsensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β 2 -adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.femtosecond diffraction | crystallography | XFEL | structural biology U sing extremely bright, short-timescale X-ray pulses produced by X-ray free-electron lasers (XFELs), femtosecond crystallography (FX) is an emerging method that expands the structural information accessible from very small or very radiation-sensitive macromolecular crystals. Central to this method is the "diffraction before destruction" (1) process in which a still diffraction image is produced by a single X-ray pulse before significant radiation-induced electronic and atomic rearrangements occur within the crystal (1-3). At the Linac Coherent Light Source (LCLS) at SLAC, a single ∼50-fs-long X-ray pulse can expose a crystal to as many X-ray photons as a typical synchrotron beam line produces in about a second. Exposing small crystals to these intense ultrashort pulses circumvents the dose limitations of conventional X-ray diffraction experiments (4) and may produce useful data to resolutions beyond what is achievable at synchrotrons (5). This innovation provides a pathway to obtain atomic information from proteins that only form micrometer-to nanometer-sized crystals, such as many membrane proteins and large multiprotein complexes. Moreover, XFELs enable "diffraction before reduction" data collection to address another major challenge in structural enzymology by providing a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzyme active sites (6), such as high-valency reaction intermediates that may be significantly photoreduced during a single X-ray exposure at a synchrotron, even at very small doses (7-11). Furthermore, the use of short (tens of femtoseconds) X-ray pulses further complements the structural characterization of biochemical reaction processes by providing access to a time domain two to three orders of magnitude faster (12, 13) than currently accessible using synchrotro...

The PTH receptor is one of the first GPCR found to sustain cAMP signaling after internalization of the ligand–receptor complex in endosomes. This unexpected model is adding a new dimension on how we think about GPCR signaling, but its mechanism is incompletely understood. We report here that endosomal acidification mediated by the PKA action on the v-ATPase provides a negative feedback mechanism by which endosomal receptor signaling is turned-off.

The current practice for identifying crystal hits for X-ray crystallography relies on optical microscopy techniques that are limited to detecting crystals no smaller than 5 μm. Because of these limitations, nanometer-sized protein crystals cannot be distinguished from common amorphous precipitates, and therefore go unnoticed during screening. These crystals would be ideal candidates for further optimization or for femtosecond X-ray protein nanocrystallography. The latter technique offers the possibility to solve high-resolution structures using submicron crystals. Transmission electron microscopy (TEM) was used to visualize nanocrystals (NCs) found in crystallization drops that would classically not be considered as "hits." We found that protein NCs were readily detected in all samples tested, including multiprotein complexes and membrane proteins. NC quality was evaluated by TEM visualization of lattices, and diffraction quality was validated by experiments in an X-ray free electron laser.T he emergence of X-ray free electron laser (X-FEL)-based serial femtosecond crystallography holds the promise of solving the 3D structure of proteins that can only crystallize as "nanocrystals" (NCs) or are highly sensitive to radiation damage (1-5). NCs appropriate for X-FEL experiments are considered to be 200 nm to 2 μm in size (6). This size is constrained primarily by the requirements of the NC delivery system to the X-FEL beam. In addition to allowing for structure resolution of NCs by X-FEL experiments, they provide the advantage of requiring no crystal cryoprotection because these experiments are performed at room temperature (3, 7). Given the opportunities that X-FELs offer to the field of crystallography, efficient methodologies to detect NCs from single crystallography drops and to optimize these identified conditions yielding NCs will be essential for future developments in structural biology. Current methods to detect the presence of NCs include dynamic light scattering (DLS), bright-field microscopy, birefringence microscopy, and intrinsic tryptophan UV fluorescence imaging, as well as technologies that rely upon second harmonic generation, such as second order nonlinear imaging of chiral crystals (SONICC) (8, 9) and X-ray powder diffraction. However, limitations of these imaging techniques include (i) ineffective detection of crystals smaller than 5 μm (8, 10), (ii) false-positive conditions as a result of interference from precipitate backgrounds (8, 10), and (iii) false-negative conditions resulting from the lack of tryptophan residues in the case of UV fluorescence and from the lack of chiral centers in the case of SONICC (11). Although DLS can accurately measure the size distribution of nanometer-sized protein aggregates, it is unable to distinguish unambiguously between amorphous and crystalline (12). Finally, X-ray powder diffraction, a method that has been applied to evaluate samples for the presence and concentration of NCs, requires more material than is produced in a single crystallization screening d...

Recent advancements at the Linac Coherent Light Source X-ray free-electron laser (XFEL) enabling successful serial femtosecond diffraction experiments using nanometre-sized crystals (NCs) have opened up the possibility of X-ray structure determination of proteins that produce only submicrometre crystals such as many membrane proteins. Careful crystal pre-characterization including compatibility testing of the sample delivery method is essential to ensure efficient use of the limited beamtime available at XFEL sources. This work demonstrates the utility of transmission electron microscopy for detecting and evaluating NCs within the carrier solutions of liquid injectors. The diffraction quality of these crystals may be assessed by examining the crystal lattice and by calculating the fast Fourier transform of the image. Injector reservoir solutions, as well as solutions collected post-injection, were evaluated for three types of protein NCs (i) the membrane protein PTHR1, (ii) the multi-protein complex Pol II-GFP and (iii) the soluble protein lysozyme. Our results indicate that the concentration and diffraction quality of NCs, particularly those with high solvent content and sensitivity to mechanical manipulation may be affected by the delivery process.

Serial femtosecond crystallography (SFX) employing high-intensity X-ray free-electron laser (XFEL) sources has enabled structural studies on microcrystalline protein samples at non-cryogenic temperatures. However, the identification and optimization of conditions that produce well diffracting microcrystals remains an experimental challenge. Here, we report parallel SFX and transmission electron microscopy (TEM) experiments using fragmented microcrystals of wild type (WT) homoprotocatechuate 2,3-dioxygenase (HPCD) and an active site variant (H200Q). Despite identical crystallization conditions and morphology, as well as similar crystal size and density, the indexing efficiency of the diffraction data collected using the H200Q variant sample was over 7-fold higher compared to the diffraction results obtained using the WT sample. TEM analysis revealed an abundance of protein aggregates, crystal conglomerates and a smaller population of highly ordered lattices in the WT sample as compared to the H200Q variant sample. While not reported herein, the 1.75 Å resolution structure of the H200Q variant was determined from ~16 minutes of beam time, demonstrating the utility of TEM analysis in evaluating sample monodispersity and lattice quality, parameters critical to the efficiency of SFX experiments.

The crystallization of protein samples remains the most significant challenge in structure determination by X-ray crystallography. Here, the effectiveness of transmission electron microscopy (TEM) analysis to aid in the crystallization of biological macromolecules is demonstrated. It was found that the presence of well ordered lattices with higher order Bragg spots, revealed by Fourier analysis of TEM images, is a good predictor of diffraction-quality crystals. Moreover, the use of TEM allowed (i) comparison of lattice quality among crystals from different conditions in crystallization screens; (ii) the detection of crystal pathologies that could contribute to poor X-ray diffraction, including crystal lattice defects, anisotropic diffraction and crystal contamination by heavy protein aggregates and nanocrystal nuclei; (iii) the qualitative estimation of crystal solvent content to explore the effect of lattice dehydration on diffraction and (iv) the selection of high-quality crystal fragments for microseeding experiments to generate reproducibly larger sized crystals. Applications to X-ray free-electron laser (XFEL) and micro-electron diffraction (microED) experiments are also discussed.