Toward G protein-coupled receptor structure-based drug design using X-ray lasers

Ishchenko, Andrii; Stauch, Benjamin; Han, Gye Won; Batyuk, A.; Shiriaeva, Anna; Li, Chufeng; Zatsepin, Nadia A.; Weierstall, Uwe; Liu, Wei; Nango, Eriko; Nakane, Takanori; Tanaka, Rie; Tono, Kensuke; Joti, Yasumasa; Iwata, So; Moraes, Isabel; Gati, Cornelius; Cherezov, Vadim

doi:10.1107/s2052252519013137

Cited by 64 publications

(55 citation statements)

References 84 publications

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“…During the last five years, about a dozen novel GPCR structures have been determined benefiting from XFEL data collection . In the case of MT receptors, using the CXI beamline at the Linac Coherent Light Source (LCLS) we were able to obtain structures of MT 1 and MT 2 bound to different agonists at resolutions between 2.8 and 3.3 Å (Table )—we attribute the remarkable gain in resolution for MT 1 at XFEL sources to the specific packing of the receptor in alternating layers with strong and weak contacts (Fig. ), as cryo‐cooling causes contraction of the crystal lattice, introducing defects in the lattice hence limiting resolution.…”

Section: Structure Determination Of Mt Receptorsmentioning

confidence: 97%

Structural insights into melatonin receptors

2019

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The long-anticipated high-resolution structures of the human melatonin G protein-coupled receptors MT 1 and MT 2 , involved in establishing and maintaining circadian rhythm, were obtained in complex with two melatonin analogs and two approved anti-insomnia and antidepression drugs using X-ray free-electron laser serial femtosecond crystallography. The structures shed light on the overall conformation and unusual structural features of melatonin receptors, as well as their ligand binding sites and the melatonergic pharmacophore, thereby providing insights into receptor subtype selectivity. The structures revealed an occluded orthosteric ligand binding site with a membrane-buried channel for ligand entry in both receptors, and an additional putative ligand entry path in MT 2 from the extracellular side. This unexpected ligand entry mode contributes to facilitating the high specificity with which melatonin receptors bind their cognate ligand and exclude structurally similar molecules such as serotonin, the biosynthetic precursor of melatonin. Finally, the MT 2 structure allowed accurate mapping of type 2 diabetes-related single-nucleotide polymorphisms, where a clustering of residues in helices I and II on the protein-membrane interface was observed which could potentially influence receptor oligomerization. The role of receptor oligomerization is further discussed in light of the differential interaction of MT 1 and MT 2 with GPR50, a regulatory melatonin coreceptor. The melatonin receptor structures will facilitate design of selective tool compounds to further dissect the specific physiological function of each receptor subtype as well as provide a structural basis for next-generation sleeping aids and other drugs targeting these receptors with higher specificity and fewer side effects.

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Section: Structure Determination Of Mt Receptorsmentioning

confidence: 97%

Structural insights into melatonin receptors

2019

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“…The combination of high flux and sub-micron focus beam (<1 µm × 1 µm) allows microcrystals (<5 µm 3 ) of angiotensin II receptor [53], δ-opioid receptor [54], CPV17 polyhedrin [55] and melatonin receptor 2 (MT2) [56] to be used for data collection, which otherwise did not provide sufficient diffraction volume at synchrotron. Serial crystallography thus represents the next frontier in X-ray crystallography and has the potential to address the crystallization bottleneck of requiring large, well-diffracting crystals of membrane proteins for SBDD [57][58][59]. A further unique potential of XFEL is the femtosecond data collection regime, allowing time-resolved dynamic studies at sub-picosecond timescales relevant to many biological processes.…”

Section: Xfel and Serial Crystallographymentioning

confidence: 99%

“…Ligand soaking into crystals in LCP has been demonstrated [216], proving that LCP retains the water channel structure which allows the efficient diffusion of small molecules and opens up the possibility of soaking experiments for samples prepared for the LCP jet. To increase throughput and collect multiple ligand complex datasets for β2 adrenergic receptor at LCLS and SACLA, protein was purified in the presence of either timolol or alprenolol and replaced with another ligand of equal or higher affinity during crystallization in LCP [59]. SFX data were then collected for each receptor/ligand complex per loading run, with data collection times between 0.5 to 3 h, depending on hit rate, XFEL repetition rate, indexing rate and the degree of data convergence (typically >10,000 indexed images) required for the purpose of the experiment [59].…”

Section: Sample Delivery Present and Futurementioning

confidence: 99%

“…To increase throughput and collect multiple ligand complex datasets for β2 adrenergic receptor at LCLS and SACLA, protein was purified in the presence of either timolol or alprenolol and replaced with another ligand of equal or higher affinity during crystallization in LCP [59]. SFX data were then collected for each receptor/ligand complex per loading run, with data collection times between 0.5 to 3 h, depending on hit rate, XFEL repetition rate, indexing rate and the degree of data convergence (typically >10,000 indexed images) required for the purpose of the experiment [59]. A similar study was performed for thermolysin [211], where crystals were first soaked with different ligands in batch before mixing into viscous medium for data collection using the LCP jet.…”

Section: Sample Delivery Present and Futurementioning

confidence: 99%

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Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

Cheng¹

2020

Crystals

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The advent of the X-ray free electron laser (XFEL) in the last decade created the discipline of serial crystallography but also the challenge of how crystal samples are delivered to X-ray. Early sample delivery methods demonstrated the proof-of-concept for serial crystallography and XFEL but were beset with challenges of high sample consumption, jet clogging and low data collection efficiency. The potential of XFEL and serial crystallography as the next frontier of structural solution by X-ray for small and weakly diffracting crystals and provision of ultra-fast time-resolved structural data spawned a huge amount of scientific interest and innovation. To utilize the full potential of XFEL and broaden its applicability to a larger variety of biological samples, researchers are challenged to develop better sample delivery methods. Thus, sample delivery is one of the key areas of research and development in the serial crystallography scientific community. Sample delivery currently falls into three main systems: jet-based methods, fixed-target chips, and drop-on-demand. Huge strides have since been made in reducing sample consumption and improving data collection efficiency, thus enabling the use of XFEL for many biological systems to provide high-resolution, radiation damage-free structural data as well as time-resolved dynamics studies. This review summarizes the current main strategies in sample delivery and their respective pros and cons, as well as some future direction.

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“…Extracting crystals from the viscous LCP is difficult, and many membrane protein crystals only grow to a few micrometers in their largest dimension. Even when relatively large crystals are available, soaking drugs into them is not always feasible, limiting SBDD applications for GPCRs (14). Therefore, structural investigations of GPCRs were greatly facilitated by the advent of X-ray free electron lasers (XFEL) with injector-based LCP delivery systems (10,15,16).…”

Section: Main Textmentioning

confidence: 99%

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

Martynowycz

Shiriaeva

et al. 2020

Preprint

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G Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods.One sentence summaryFIB milled LCP-GPCR structure determined by MicroED

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Toward G protein-coupled receptor structure-based drug design using X-ray lasers

Abstract: A method is presented for efficient co-crystal structure determination for G protein-coupled receptors taking advantage of serial femtosecond crystallography.

Cited by 64 publications

References 84 publications

Structural insights into melatonin receptors

Structural insights into melatonin receptors

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP

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