New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

Ratnala, Venkata R. P.

doi:10.1007/s10529-006-9005-y

Cited by 14 publications

(9 citation statements)

References 60 publications

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“…Taking advantage of a concerted action of CHS and a high affinity ligand the CB 2 receptor stabilized in either ground (SR-144,528) or activated (CP-55,940) functional conformations can be prepared. Stabilization by the high affinity ligands was previously reported for other GPCRs including β2 adrenergic receptor [45], CXCR4/δ opioid receptor [46] and others [47].…”

Section: Discussionmentioning

confidence: 57%

Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers

et al. 2012

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Elucidation of the molecular mechanisms of activation of G protein-coupled receptors (GPCRs) is among the most challenging tasks for modern membrane biology. For studies by high resolution analytical methods, these integral membrane receptors have to be expressed in large quantities, solubilized from cell membranes and purified in detergent micelles, which may result in a severe destabilization and a loss of function. Here, we report insights into differential effects of detergents, lipids and cannabinoid ligands on stability of the recombinant cannabinoid receptor CB2, and provide guidelines for preparation and handling of the fully functional receptor suitable for a wide array of downstream applications. While we previously described the expression in Escherichia coli, purification and liposome-reconstitution of multi-milligram quantities of CB2, here we report an efficient stabilization of the recombinant receptor in micelles - crucial for functional and structural characterization. The effects of detergents, lipids and specific ligands on structural stability of CB2 were assessed by studying activation of G proteins by the purified receptor reconstituted into liposomes. Functional structure of the ligand binding pocket of the receptor was confirmed by binding of 2H-labeled ligand measured by solid-state NMR. We demonstrate that a concerted action of an anionic cholesterol derivative, cholesteryl hemisuccinate (CHS) and high affinity cannabinoid ligands CP-55,940 or SR-144,528 are required for efficient stabilization of the functional fold of CB2 in dodecyl maltoside (DDM)/CHAPS detergent solutions. Similar to CHS, the negatively charged phospholipids with the serine headgroup (PS) exerted significant stabilizing effects in micelles while uncharged phospholipids were not effective. The purified CB2 reconstituted into lipid bilayers retained functionality for up to several weeks enabling high resolution structural studies of this GPCR at physiologically relevant conditions.

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Section: Discussionmentioning

confidence: 57%

Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers

et al. 2012

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“…However, the complexity of the solid-state NMR spectra and difficulties related to sample preparation have prevented, so far, the solution of the structure of whole GPCRs through NMR. For these reasons, most of the studies have been directed toward the solution of individual portions of the receptor structure either in aqueous solution or in different lipid-mimetic solvents [41]. In Table 1, we report a synoptic view of the available 3D structural data.…”

Section: Determination Of the 3d Structure Of Gpcrsmentioning

confidence: 99%

“…The methods and technical challenges underlying the application of NMR spectroscopy to membrane proteins and GPCRs have been covered in recently published reviews [41–43]. Here, we will focus on the advances in the understanding of the structure and function of GPCRs that have been made through NMR studies.…”

mentioning

confidence: 99%

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

Tikhonova¹,

Costanzi²

2009

CPD

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G protein-coupled receptors (GPCRs) are a large superfamily of signaling proteins expressed on the plasma membrane. They are involved in a wide range of physiological processes and, therefore, are exploited as drug targets in a multitude of therapeutic areas. In this extent, knowledge of structural and functional properties of GPCRs may greatly facilitate rational design of modulator compounds. Solution and solid-state nuclear magnetic resonance (NMR) spectroscopy represents a powerful method to gather atomistic insights into protein structure and dynamics. In spite of the difficulties inherent the solution of the structure of membrane proteins through NMR, these methods have been successfully applied, sometimes in combination with molecular modeling, to the determination of the structure of GPCR fragments, the mapping of receptor-ligand interactions, and the study of the conformational changes associated with the activation of the receptors. In this review, we provide a summary of the NMR contributions to the study of the structure and function of GPCRs, also in light of the published crystal structures. KeywordsG protein-coupled receptors (GPCRs); NMR; three-dimensional structures; ligand recognition; receptor activation G protein-coupled receptors (GPCRs), also known as seven transmembrane-spanning receptors (7TMRs), are a large superfamily of signaling proteins expressed on the plasma membrane that function as receivers for extracellular stimuli [1]. About 1000 GPCRs have been identified in the human genome [2]. Since their signaling is involved in numerous physiological functions and pathological conditions, GPCRs constitute the molecular target of a significant percentage of the currently marketed drugs. Moreover, many additional members of the superfamily have been identified as potential targets for the treatment of a variety of diseases and are the object of substantial drug discovery efforts. From the molecular point of view, all GPCRs share a common molecular architecture, being composed of a single polypeptide chain folded into a bundle of seven α-helical transmembrane domains (TMs) connected by three extracellular and three intracellular loops (ELs and ILs). The N-terminus is located in the extracellular space, while the C-terminus is in the cytosol (Fig. (1)).Given that structure-based drug discovery is an efficient method to rationally design novel drugs and improve the properties of old drugs, the scientific community has been striving for a long time to shed light onto the elusive structure-function relationships of GPCRs employing a variety of direct biophysical and indirect biochemical methods [3]. The most direct method that has been used is X-ray crystallography. However, up until now, this technique has been *address for correspondence: stefanoc@mail.nih.gov. NIH Public Access Author ManuscriptCurr Pharm Des. Author manuscript; available in PMC 2010 January 5. Published in final edited form as:Curr Pharm Des. 2009 ; 15(35): 4003-4016. NIH-PA Author ManuscriptNIH-PA Author Manuscript...

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“…The prospects for elucidating the structures of other GPCR are not very high, and await a major breakthrough (45,46). Structural information on GPCRs could be attained by techniques of electron crystallography, electron paramagnetic resonance, UV absorbance and fluorescence spectroscopy, nuclear magnetic resonance (NMR) spectroscopy (47)(48)(49) as well as computer modeling. The predicted structures could also be validated by some other experimental techniques such as substituted cysteine accessibility method (SCAM) (50,51) and site directed mutagenesis (52,53).…”

Section: Gpcrs and Their Structure Modeling Techniquesmentioning

confidence: 99%

“…The rhodopsin crystal structure has often been used as a template in the homology modeling of the transmembrane region of several GPCR subtypes. This paradigm is based on experimental evidence that part of the template conformation is similar to other GPCRs (29,38,47,49,55).…”

Section: De Novo and Ab Initio Modelsmentioning

confidence: 99%

Computerized modeling techniques predict the 3D structure of H4R: Facts and fiction

Zaid

Ismael-Shanak²,

Michaeli³

et al. 2012

Front Biosci

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The functional characterization of proteins presents a daily challenge r biochemical, medical and computational sciences, especially when the structures are undetermined empirically, as in the case of the Histamine H4 Receptor (H₄R). H₄R is a member of the GPCR superfamily that plays a vital role in immune and inflammatory responses. To date, the concept of GPCRs modeling is highlighted in textbooks and pharmaceutical pamphlets, and this group of proteins has been the subject of almost 3500 publications in the scientific literature. The dynamic nature of determining the GPCRs structure was elucidated through elegant and creative modeling methodologies, implemented by many groups around the world. H₄R which belongs to the GPCR family was cloned in 2000; understandably, its biological activity was reported only 65 times in pubmed. Here we attempt to cover the fundamental concepts of H₄R structure modeling and its implementation in drug discovery, especially those that have been experimentally tested and to highlight some ideas that are currently being discussed on the dynamic nature of H₄R and GPCRs computerized techniques for 3D structure modeling.

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New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

Cited by 14 publications

References 60 publications

Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers

Stabilization of Functional Recombinant Cannabinoid Receptor CB2 in Detergent Micelles and Lipid Bilayers

Unraveling the Structure and Function of G Protein-Coupled Receptors Through NMR Spectroscopy

Computerized modeling techniques predict the 3D structure of H4R: Facts and fiction

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