We have recently established a procedure for serial femtosecond crystallography (SFX) in lipidic cubic phase (LCP) for protein structure determination at X-ray free-electron lasers (XFELs). LCP-SFX ...uses the gel-like LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room-temperature high-resolution data with minimal effects of radiation damage from sub-10-μm crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared with SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2-3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3-5 d, including the time required for the crystals to grow.
Metabotropic γ-aminobutyric acid receptors (GABA
) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that ...range from addiction to psychosis
. GABA
belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits
. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA
. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.
The angiotensin II receptors AT
R and AT
R serve as key components of the renin-angiotensin-aldosterone system. AT
R has a central role in the regulation of blood pressure, but the function of AT
R ...is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT
R bound to an AT
R-selective ligand and to an AT
R/AT
R dual ligand, 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 or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands.
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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•Crystal structure of the human Angiotensin II type 1 receptor at 2.9-Å resolution•Structure is solved by X-ray laser serial femtosecond crystallography•Antagonist ZD7155 forms critical interactions with Tyr35, Trp84 and Arg167•Docking reveals binding modes of common angiotensin receptor blockers
Structure determination of human Angiotensin II type 1 receptor bound to an antagonist using serial femtosecond crystallography with X-ray free-electron laser and docking studies of other common anti-hypertensive drugs into the structure offer insights into design of blood pressure modulators.
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.
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the ...regulation of blood pressure and core body temperature, oncogenesis, and immune function. Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness by activating two high-affinity G-proteincoupled receptors, type 1A (MT1) and type 1B (MT2). Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids, and is one of the most popular supplements in the United States. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine. The structure of MT2 is described in an accompanying paper. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.
Abstract
Despite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A ...major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.
Cysteinyl leukotriene G protein-coupled receptors CysLT
and CysLT
regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT
R has been used for ...treating asthma and associated diseases for over two decades, CysLT
R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT
R in complex with three dual CysLT
R/CysLT
R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants.
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the ...crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.
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