A transmembrane helix‐bundle from G‐protein coupled receptor CB2: Biosynthesis, purification, and NMR characterization

Zheng, Haian; Zhao, Jun; Sheng, Wanyun; Xie, Xiang‐Qun

doi:10.1002/bip.20526

Cited by 28 publications

(34 citation statements)

References 59 publications

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“…A large body of literature supports the basic assumption of the model: For example, proteolysis of membrane proteins resulted in fragments containing entire TM sequences (Huang et al 1981), and chemically or recombinantly synthesized TM peptides spontaneously assembled thereby rescuing receptor activity (Kahn and Engelman 1992;Ridge et al 1995;Martin et al 1999;Wrubel et al 1994). Finally, peptides corresponding to the N and C terminus (Harmar 2001;O'Hara et al 1993), loop domains (Bennett et al 2004;Katragadda et al 2001a, b;Yeagle et al 2000) and transmembrane domains (Katragadda et al 2001a, b;Cohen et al 2008;Zheng et al 2006;Musial-Siwek et al 2008;Tian et al 2007;Lau et al 2008;Mobley et al 2007;Neumoin et al 2007) from GPCRs have been found to fold to distinct secondary structures which in certain cases resembled the structures of the corresponding regions of the intact receptor.…”

Section: Introductionmentioning

confidence: 67%

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

2008

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The human Y4 receptor, a class A G-protein coupled receptor (GPCR) primarily targeted by the pancreatic polypeptide (PP), is involved in a large number of physiologically important functions. This paper investigates a Y4 receptor fragment (N-TM1-TM2) comprising the N-terminal domain, the first two transmembrane (TM) helices and the first extracellular loop followed by a (His) 6 tag, and addresses synthetic problems encountered when recombinantly producing such fragments from GPCRs in Escherichia coli. Rigorous purification and usage of the optimized detergent mixture 28 mM dodecylphosphocholine (DPC)/118 mM% 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) resulted in high quality TROSY spectra indicating protein conformational homogeneity. Almost complete assignment of the backbone, including all TM residue resonances was obtained. Data on internal backbone dynamics revealed a high secondary structure content for N-TM1-TM2. Secondary chemical shifts and sequential amide proton nuclear Overhauser effects defined the TM helices. Interestingly, the properties of the N-terminal domain of this large fragment are highly similar to those determined on the isolated N-terminal domain in the presence of DPC micelles.

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Section: Introductionmentioning

confidence: 67%

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

2008

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“…Probably, even greater insights could be obtained from the analyses of larger substructures or even whole receptors. At this purpose, Zheng and coworkers biosynthesized a quite large segment of the cannabinoid CB 2 receptor, including TM1, IL1 and TM2, using a fusion protein overexpression strategy [50]. The preliminary results gathered by the authors for the 13 C/ 15 N labeled peptide argue in favor of the applicability of this strategy to the synthesis of GPCR helical bundles for NMR studies.…”

Section: Determination Of the 3d Structure Of Gpcrsmentioning

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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“…We previously showed high level expression of fusion proteins when TrpLE was fused to the N terminal of other CB2 fragments [12,14,15]. In the current study, the human CB2 271-326 fragment was fused with TrpLE and the fusion protein was exclusively achieved in the form of IBs in E. coli cells.…”

Section: Resultsmentioning

confidence: 72%

“…Our lab has previously over-expressed, purified and structurally characterized CB2 fragments, including CB2 180-233 , CB2 , and CB2 , which correspond to TM1-IL1-TM2 (from the first transmembrane domain to the second intracellular loop and the second transmembrane domain), TM2 and TM5-IL3 [12][13][14][15] respectively. In these publications, the CB2 fragments were expressed as fusion proteins with a modified TrpΔLE1413 (TrpLE) leader sequence and a 9-histidine tag at the N-terminus.…”

mentioning

confidence: 99%

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

Zhang

Xie

2008

Protein Expression and Purification

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Obtaining sufficient amount of purified G-protein coupled receptors (GPCRs) is almost always one of the major challenges for their structural studies. CB2 , a human cannabinoid receptor 2 (CB2) fragment comprising part of the third extracellular loop (EL3), the seventh transmembrane domain (TM7) and C-terminal juxtamembrane region of the receptor, was over-expressed as a fusion protein into inclusion body (IB) of Escherichia coli. The fusion protein was purified by histidineselected nickel affinity chromatography under denaturing conditions. Then, the fusion protein IBs were solubilized in detergent (Brij58) and the expression fusion leader sequence (TrpLE) was specifically cleaved with tobacco etch virus (TEV) protease. The target fragment, CB2 , was subsequently purified by reverse-phase HPLC and confirmed by SDS-PAGE and mass spectrometry. This hydrophobic fragment can refold in mild detergents digitonin and Brij58. Circular dichroism (CD) spectroscopy of CB2 in digitonin and Brij58 micelles showed that the fragment adopts a more than 75% α-helical structure, with the remainder having β-strand structure. Fluorescence spectroscopy and quenching studies suggested that the C-terminal region lies near the surface of the digitonin micelles and the TM7 region is folded relatively close to the center of the micelles. This study may provide an alternative strategy for the production and structure/functional studies of GPCRs such as CB2 receptor protein produced in the form of IBs. KeywordsCannabinoid receptor 2; transmembrane domain; inclusion body; affinity chromatography; HPLC; circular dichroism; fluorescence Since the discovery of the CB2 receptor in 1993 [1], there has been a growing interest to clarify the importance of this GPCR membrane protein in human physiology, and to investigate it as a possible target for current and future drug development. The CB2 receptor seems to have a significant role in mediating the immunological functions of cannabinoid receptor ligands. Thus, the CB2 receptor is a promising target for drug development that aims at management of neurological pain, inflammation, osteoporosis, and growth of malignant gliomas and tumors * Corresponding author. Fax: +1 412 383 5276. E-mail address: xix15@pitt.edu (X-Q. Xie). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][4][5][6][7]. In general, the Escherichia coli expression system is the most attractive strategy due to its advantages of simplicity of use, low cost, easy scale-up, and homogeneous protein production. Though few attempts were promising to express full length CB2 receptor in ...

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A transmembrane helix‐bundle from G‐protein coupled receptor CB2: Biosynthesis, purification, and NMR characterization

Cited by 28 publications

References 59 publications

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

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

Biosynthesis, purification, and characterization of a cannabinoid receptor 2 fragment (CB2271–326)

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