Towards high throughput GPCR crystallography: In Meso soaking of Adenosine A2A Receptor crystals

Rucktooa, Prakash; Cheng, R.K.; Segala, Elena; Geng, Tao; Errey, James C.; Brown, Giles H.; Cooke, Robert M.; Marshall, Fiona H.; Dore, A.S.

doi:10.1038/s41598-017-18570-w

Cited by 86 publications

(71 citation statements)

References 36 publications

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“…However, once a crystal structure has been determined, they can often attain much higher resolution than structures of membrane proteins obtained so far, although high resolution cryo-EM structures are possible from single molecule imaging 34 . Another current advantage of X-ray crystallography is the possibility of soaking crystals to get multiple structures of a receptor bound to different ligands through molecular replacement 35 . Finally, there is still a size limitation of the molecule imaged by cryo-EM for structure determination 36 and experimentally this is now at about 65 kDa 25 .…”

Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Tate

García-Nafría

Lee

et al. 2018

Preprint

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34The adenosine A 2A receptor (A 2A R) is a prototypical G protein-coupled receptor (GPCR) that 35 couples to the heterotrimeric G protein G S . Here we determine the structure by electron cryo-36 microscopy (cryo-EM) of A 2A R at pH 7.5 bound to the small molecule agonist NECA and 37 coupled to an engineered heterotrimeric G protein, which contains mini-G S , the βγ subunits 38 and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. 39 Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-G S 40 are virtually identical and that the density of the side chains and ligand are of comparable 41 quality. However, the cryo-EM density map also indicates regions that are flexible in 42 comparison to the crystal structures, which unexpectedly includes regions in the ligand 43 binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the 44 β subunit of the G protein was observed. 45 46 47 48 49The adenosine A 2A receptor (A 2A R) is an archetypical Class A G protein-coupled 50 receptor (GPCR) 1 . A 2A R is activated by the endogenous agonist adenosine and plays a 51 prominent role in cardiac function, the immune system and central nervous system, including 52 the release of the major excitatory neurotransmitter glutamate 2,3 . Given the widespread tissue 53 distribution and physiological relevance of A 2A R, it is a validated drug target for many 54 disorders 4 , including Parkinson's disease 5 and cancer 6 . A 2A R is one of the most stable GPCRs 55 and structures have been determined of A 2A R in an inactive state bound to inverse agonists 7-56 13 , an active intermediate state bound to agonists 14-16 and the fully active state bound to an 57 agonist and coupled to an engineered G protein, mini-G S 17 . In addition, structure-based drug 58 design has been applied to inactive state structures of A 2A R to develop potent and subtype 59 specific inverse agonists with novel scaffolds 9 and these are currently in clinical trials. 60Comparison of the structures has led to an understanding of the molecular determinants for 61 an inverse agonist compared to an agonist 15 , the conformational changes induced by agonist 62 binding to convert the inactive state to the active intermediate state 18 , and the role of the G 63 protein in stabilising the fully active state 17 . The active state was determined by crystallizing 64 the receptor coupled solely to mini-G S , an engineered G protein with eight point mutations 65 and three deletions, including the whole of the α-helical domain 19 . Although 66 pharmacologically mini-G S recapitulates the ability of a heterotrimeric G protein to increase 67 the affinity of agonist binding to the receptor 17 , the roles for the βγ subunits could not be 68 described. In terms of the interactions between a heterotrimeric G protein and a Class A 69 GPCR, the vast majority of interactions are made by the α subunit, in particular the C-70 terminal α5 helix 20 . However, there was an interaction be...

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Section: Discussionmentioning

confidence: 99%

Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Tate

García-Nafría

Lee

et al. 2018

Preprint

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“…Jet-based sample delivery methods present severe hurdles to this, as only one sample can be investigated per loading and each sample changing still incurs a significant time penalty during a beamtime run. 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].…”

Section: Sample Delivery Present and Futurementioning

confidence: 99%

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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“…The structures of the human A2A adenosine receptor in complexes with the chromones were obtained by following experimental protocols described previously. 31,32 Briefly, the A2A receptor expression construct has been extensively engineered, and features truncations, thermostabilising mutations, and is fused to the Apocytochrome bRIL562 in ICL3, and to a decahistidine tag at the C-terminus. The construct was expressed using the Bac to Bac Expression System (Invitrogen) in Trichoplusa ni Tni PRO cells, and the protein was purified at 4°C in decyl-β-maltopyranoside, and in the presence of 1mM of Theophylline, using metal affinity (Ni-NTA Superflow, QIAGEN) and size exclusion chromatography (Super-dex200, GE Healthcare).…”

Section: N Conclusionmentioning

confidence: 99%

“…The final data reduction statistics are presented in Supporting Information Table S2. The structures of the A2A-StaR2-bRIL562-chromone complexes were solved by molecular replacement (MR) with Phaser 64 using the previously reported A2A-StaR2-bRIL562theophylline complex structure 31 as the search model (PDB code: 5MZJ). Model refinement was performed using phenix.refine, 65 including TLS refinement for 2 groups corresponding to the receptor and bRIL562 respectively.…”

Section: N Conclusionmentioning

confidence: 99%

“…In this case, the starting configuration of the complex was a result of cross docking this ligand, co-crystallized with the STAR version of the A2AAR (3UZA), which presents a binding mode conserved other triazine structures ( Figure 1B). 31 This protocol allows to revert the stabilizing mutations of the STAR receptor in a unified A2AAR receptor structure, which is used along the FEP explorations in this study. The predicted values (∆∆ #$%& 9:;9 ) show comparable statistical values as in the case of ZM241385 (MAE = 0.94 kcal/mol; R 2 = 0.66).…”

Section: N Introductionmentioning

confidence: 99%

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X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists

Jespers¹,

Verdon²,

Azuaje³

et al. 2019

Preprint

Self Cite

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<div> <div> <div> <p>Nowadays, rigorous free energy calculations are routinely considered in pharmaceutical design strategies. One typical sce- nario is the lead-optimization based on well-defined protein-ligand binding modes, inferred by pharmacological data in com- putational models and ultimately revealed by structural data. In this work, we reveal the molecular determinants of antago- nist binding to the adenosine A2A adenosine receptor (AR), an emerging target in immuno-oncology, via a robust protocol that connects structural and pharmacological data through free energy perturbation (FEP) calculations. Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with FEP simulations of each side-chain mutation, performed on alternate binding modes previously proposed in the literature. The results strongly suggested that only one binding mode could explain this experimental data, which was used to subsequently design a series of 11 chromone deriva- tives. The experimental affinities of these new compounds were linked through a cycle of ligand-FEP calculations around selected ligand pairs, which allowed the identification of the optimal positioning of the different chemical substituents in the proposed binding model. Subsequent X-ray crystallography of the A2AAR with a low and high affinity chromone derivative confirmed the predicted binding orientation, and provided new insights in the role of the explored substituents in the chro- </p> </div> </div> <div> <div> <p>mone scaffold. </p> </div> </div> </div>

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Towards high throughput GPCR crystallography: In Meso soaking of Adenosine A2A Receptor crystals

Cited by 86 publications

References 36 publications

Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists

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Towards high throughput GPCR crystallography: In Meso soaking of Adenosine A2A Receptor crystals

Cited by 86 publications

References 36 publications

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

X-Ray Crystallography and Free Energy Calculations Reveal the Binding Mechanism of A2A Adenosine Receptor Antagonists

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Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein

Cryo-EM structure of the adenosine A_2A receptor coupled to an engineered heterotrimeric G protein