Generating thermostabilized agonist-bound GPR40/FFAR1 using virus-like particles and a label-free binding assay

Abstract

Elucidating the detailed mechanism of activation of membrane protein receptors and their ligand binding is essential for structure-based drug design. Membrane protein crystal structure analysis successfully aids in understanding these fundamental molecular interactions. However, protein crystal structure analysis of the G-protein-coupled receptor (GPCR) remains challenging, even for the class of GPCRs which have been included in the majority of structure analysis reports among membrane proteins, due to the substantial instability of these receptors when extracted from lipid bilayer membranes. It is known that increased thermostability tends to decrease conformational flexibility, which contributes to the generation of diffraction quality crystals. However, this is still not straightforward, and significant effort is required to identify thermostabilized mutants that are optimal for crystallography. To address this issue, a versatile screening platform based on a label-free ligand binding assay combined with transient overexpression in virus-like particles was developed. This platform was used to generate thermostabilized GPR40 [also known as free fatty acid receptor 1 (FFAR1)] for fasiglifam (TAK-875). This demonstrated that the thermostabilized mutant GPR40 (L42A/F88A/G103A/Y202F) was successfully used for crystal structure analysis.

Micro‐scale and rapid expression screening of highly expressed and/or stable membrane protein variants in Saccharomyces cerevisiae

“…However, for many membrane proteins, it is not easy to obtain suitable labeled ligands. Screening of stabilized GPCR mutants using the size‐exclusion chromatography/liquid chromatography mass spectroscopy (SEC/LC‐MS)‐based ligand binding assay was recently reported; this protocol enabled a label‐free ligand binding assay . Since LC‐MS requires relatively costly instrumentation, it might be difficult for most researchers to utilize this technique for routine screening.…”

“…Screening of stabilized GPCR mutants using the size-exclusion chromatography/liquid chromatography mass spectroscopy (SEC/ LC-MS)-based ligand binding assay was recently reported; this protocol enabled a label-free ligand binding assay. 42 Since LC-MS requires relatively costly instrumentation, it might be difficult for most researchers to utilize this technique for routine screening. In contrast, in the screening system developed herein, the stability of the membrane protein could be evaluated by FSEC, and the inactivated receptor could be distinguished also by FSEC; therefore, this method can be implemented by using conventional HPLC.…”

Stabilization of G protein-coupled receptors by point mutations

“…While there is a set of commonly used fusion partners available for stabilization of GPCRs, a new set of stabilizing mutations had to be identified for each receptor so far. Stabilizing mutations have been found using a variety of techniques including random error-prone PCR or all-vs.-all mutation combined with evolutionary approaches (Sarkar et al, 2008 ; Dodevski and Plückthun, 2011 ; Schlinkmann and Plückthun, 2013 ; Scott and Plückthun, 2013 ; Scott et al, 2014 ) and systematic alanine scanning mutagenesis (Magnani et al, 2008 ; Serrano-Vega et al, 2008 ; Shibata et al, 2009 ; Robertson et al, 2011 ; Hollenstein et al, 2013 ; Doré et al, 2014 ; Hirozane et al, 2014 ). Most commonly used assays for conformational, thermal and detergent stability include radio-ligand binding assays, fluorescence size-exclusion chromatography (FSEC) and fluorescence activated cell sorting (FACS) with fluorescently-labeled ligands.…”

Large-scale production and protein engineering of G protein-coupled receptors for structural studies

“…A very successful approach for increasing thermostability of GPCRs was a systematic alanine scanning mutagenesis where each amino acid residue was mutated to alanine (or to leucine, if it had already been alanine). It was applied for turkey β 1 -adrenoceptor ( Serrano-Vega et al, 2008 ; Warne et al, 2009 ), human A 2A receptor stabilized both in the antagonist ( Magnani et al, 2008 ; Doré et al, 2011 ) and agonist ( Lebon et al, 2011a , b ) binding conformations, NTS 1 receptor ( Shibata et al, 2009 ; White et al, 2012 ), CRF 1 receptor ( Hollenstein et al, 2013 ) and, most recently, for mGlu 5 ( Doré et al, 2014 ) and FFA1 receptor ( Hirozane et al, 2014 ; Srivastava et al, 2014 ). Following expression, either in E. coli or in transiently transfected HEK293T cells, each mutant was solubilized in detergent solution and tested for thermostability by a radioligand binding assay.…”

“…Following expression, either in E. coli or in transiently transfected HEK293T cells, each mutant was solubilized in detergent solution and tested for thermostability by a radioligand binding assay. The only exception to this approach is FFA1 receptor ( Hirozane et al, 2014 ). Mutants of FFA1 receptor were expressed in human FreeStyle293 (Life Technologies) cells following transient cotransfection with mammalian virus-like particles and the thermostabilizing mutations were identified in a binding assay based on SEC coupled with liquid chromatography–mass spectroscopy (SEC/LC-MS).…”