G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G proteinindependent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms § Correspondence to H. Eric Xu: Eric.Xu@vai.org. * These authors contributed equally.Contributions: Y.K. initiated the project, developed the expression and purification methods for rhodopsin-arrestin complex, and bulk-purified expression constructs and proteins used in LCP crystallization for the SFX method; X.E.Z. collected the synchrotron data, helped with the SFX data collection, processed the data, and solved the structures; X.G. expressed and purified rhodopsinarrestin complexes, characterized their binding and thermal stability, discovered the initial crystallization conditions with 9.7 MAG, prepared most crystals for synchrotron data collection, prepared all crystals for the final data collection by SFX, helped with SFX data collection, and established the initial cross-linking method for the rhodopsin-arrestin complex; Y.H. designed and performed Tango assays and disulfide bond cross-linking experiments; C.Z. developed the mammalian expression methods; P.W.dW helped with XFEL data processing and performed computational experiments; J.K., M.H.E.T., K. M. S-P., K. P., J. M., Y.J., X.Y.Z., and Q.C. performed cell culture, mutagenesis, protein purification, rhodopsin-arrestin binding experiments; W.L. and A.I. grew crystals and collected synchrotron data at APS and SFX data at LCLS, G.W.H. and Q.X. determined and validated the structure. Z.Z. and V.K. constructed the full model, the phosphorylated rhodopsin-arrestin model, and help writing the paper; D.W., S.L., D.J., C.K., Sh.B., and N.A. Z. helped with XFEL data collection and initial data analysis; S.B., M.M., and G.J.W. set up the XFEL experiment, performed the data collection, and commented on the paper. A.B., T.W., C.G., O.Y., and H.C. helped with XFEL data collection and data analysis, processed the data and helped with structure validation. G.M. W., B.P., and P.G. performed HDX experiments and helped with manuscript writing. J.L. helped initiate this collaborative project and with writing the paper. M.W. collected the 7.7 Å dataset at Swiss Light Source. A.M.,...

SUMMARY Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 Å resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.

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One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the nitrate/nitrite sensor histidine kinase NarQ in the ligand-bound and mutated ligand-free states. The structures reveal that the ligand binding induces rearrangements and pistonlike shifts of TM helices. The HAMP domain protomers undergo leverlike motions and convert these pistonlike motions into helical rotations. Our findings provide the structural framework for complete understanding of TM TCS signaling and for development of antimicrobial treatments targeting TCSs.

Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt(1)-Tic(2)-Phe(3)-Phe(4)-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt(1) and Tic(2). The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.

Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands.

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and are implicated in various neuropsychological conditions, ranging from addiction to psychosis 1 . GABAB belongs to G protein-coupled receptor class C, and its functional entity consists of an obligate heterodimer composed of GB1 and GB2 2 .Each subunit possesses an extracellular Venus flytrap domain, connected to a canonical seventransmembrane domain. Here, we present four cryo-EM structures of the human full-length GB1-GB2 heterodimer in its inactive apo, two intermediate agonist-bound, and active agonist/positive allosteric modulator bound forms. The structures reveal startling differences, shedding light onto the complex motions underlying the unique activation mechanism of GABAB. Our results show that agonist binding in the GB1 Venus flytrap domain triggers a series of transitions, first bringing the two transmembrane domains into contact and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism, potentiated by a positive allosteric modulator binding at the dimerization interface, to initiate downstream signaling.

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 mm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A 2A adenosine receptor (A 2A AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX datareduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A 2A AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A 2A AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 mm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.