Fast iodide-SAD phasing for high-throughput membrane protein structure determination

Melnikov, I. A.; Polovinkin, Vitaly; Kovalev, Kirill; Gushchin, Ivan; Shevtsov, M.B.; Shevchenko, Vitaly; Mishin, Alexey; Alekseev, Alexey; Rodrı́guez-Valera, Francisco; Borshchevskiy, Valentin; Cherezov, Vadim; Leonard, Gordon A.; Gordeliy, Valentin; Popov, A.

doi:10.1126/sciadv.1602952

Cited by 40 publications

(36 citation statements)

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“…This software has already been installed at the ESRF MX beamlines and used within the context of the SSX BAG for one year. Successful applications have already been published (Zander et al, 2015(Zander et al, , 2016Melnikov et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Hierarchical clustering for multiple-crystal macromolecular crystallography experiments: the ccCluster program

et al. 2017

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This article describes ccCluster, a software providing an intuitive graphical user interface (GUI) and multiple functions to perform hierarchical cluster analysis on multiple crystallographic datasets. The program makes it easier for users to choose, in the case of multi-crystal data collection, those datasets that will be merged together to give good final statistics. It provides a simple GUI to analyse the dendrogram and various options for automated clustering and data merging.

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Section: Discussionmentioning

confidence: 99%

Hierarchical clustering for multiple-crystal macromolecular crystallography experiments: the ccCluster program

et al. 2017

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“…This structure was facilitated by a combined approach of using a high affinity ligand, which locks the receptor in its inactive state, and by replacing the third intracellular loop (ICL3) with a T4 lysozyme (T4L) fusion protein (Rosenbaum et al, 2007 ), which increases the hydrophilic surface area available for crystal contact formation (Figure 1A ). This fusion protein strategy was subsequently modified to utilize apocytochrome b 562 RIL (BRIL) instead of T4L (Liu et al, 2012 ), which resulted in the solution of seven additional structures of A 2A R bound to ZM241385 ranging in resolution from 3.2 to 1.8 Å (Liu et al, 2012 ; Batyuk et al, 2016 ; Martin-Garcia et al, 2017 ; Melnikov et al, 2017 ), and one structure bound to the antagonist 8D1 (Sun et al, 2017 ) (Table 1 ). Conformational thermostabilization, which utilizes alanine scanning mutagenesis to identify point mutations that stabilize the receptor in a particular conformational state and increase its thermostability in detergent (Magnani et al, 2008 , 2016 ), was also applied to solve the structure of A 2A R bound to ZM241385 (Doré et al, 2011 ).…”

Section: A 2a R Crystallization: Selection Of Diffmentioning

confidence: 99%

Human Adenosine A2A Receptor: Molecular Mechanism of Ligand Binding and Activation

Carpenter

Lebon

2017

Front. Pharmacol.

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Adenosine receptors (ARs) comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs). ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR), making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.

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“…These studies show that, upon the addition of a full agonist, A2aR activation follows outward movements of TM5 and TM6 (including rotation in the latter), an inward shift of the intracellular part of TM7, and a vertical translation of TM3 [42,[61][62][63][64]. Also, A2aR has received a lot of attention regarding ligand binding, lipid allosteric modulation and its activation process in MD simulations [44,[65][66][67][68][69][70][71][72][73][74][75][76][77][78] partly because it is a receptor that has been crystallized in three distinct conformational states: inactive (in the presence of an antagonist or inverse agonist) [29,63,[79][80][81][82][83][84][85][86][87][88][89][90][91], intermediate (in the presence of an agonist) [92][93][94][95], and active (in the presence of an agonist plus modified or native stimulatory G proteins) [24,25] (S1 Table).…”

Section: Introductionmentioning

confidence: 99%

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

2020

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The activation process of G protein-coupled receptors (GPCRs) has been extensively studied, both experimentally and computationally. In particular, Molecular Dynamics (MD) simulations have proven useful in exploring GPCR conformational space. The typical behaviour of class A GPCRs, when subjected to unbiased MD simulations from their crystallized inactive state, is to fluctuate between inactive and intermediate(s) conformations, even with bound agonist. Fully active conformation(s) are rarely stabilized unless a G protein is also bound. Despite several crystal structures of the adenosine A2a receptor (A2aR) having been resolved in complex with co-crystallized agonists and G s protein, its agonist-mediated activation process is still not completely understood. In order to thoroughly examine the conformational landscape of A2aR activation, we performed unbiased microsecond-length MD simulations in quadruplicate, starting from the inactive conformation either in apo or with bound agonists: endogenous adenosine or synthetic NECA, embedded in two homogeneous phospholipid membranes: 1,2-dioleoyl-sn-glycerol-3-phosphoglycerol (DOPG) or 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). In DOPC with bound adenosine or NECA, we observe transition to an intermediate receptor conformation consistent with the known adenosine-bound crystal state. In apo state in DOPG, two different intermediate conformations are obtained. One is similar to that observed with bound adenosine in DOPC, while the other is closer to the active state but not yet fully active. Exclusively, in DOPG with bound adenosine or NECA, we reproducibly identify receptor conformations with fully active features, which are able to dock G s protein. These different receptor conformations can be attributed to the action/absence of agonist and phospholipid-mediated allosteric effects on the intracellular side of the receptor.

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Fast iodide-SAD phasing for high-throughput membrane protein structure determination

Abstract: A potentially universal method for the de novo solution of the crystal structures of membrane proteins is described.

Cited by 40 publications

References 43 publications

Hierarchical clustering for multiple-crystal macromolecular crystallography experiments: the ccCluster program

Hierarchical clustering for multiple-crystal macromolecular crystallography experiments: the ccCluster program

Human Adenosine A2A Receptor: Molecular Mechanism of Ligand Binding and Activation

Insights into adenosine A2A receptor activation through cooperative modulation of agonist and allosteric lipid interactions

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