A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution.


Abstract

Structural and biophysical studies as well as drug screening approaches on G protein-coupled receptors (GPCRs) have been largely hampered by the poor biophysical properties and low expression yields of this largest class of integral membrane proteins. Thermostabilisation of GPCRs by introduction of stabilising mutations has been a key factor to overcome these limitations. However, labelled ligands with sufficient affinity, which are required for selective binding to the correctly folded receptor, are often not available. Here we describe a novel procedure to improve receptor expression and stability in a generic way, independent of specific ligands, by means of directed evolution in E. coli. We have engineered a homogenous fluorescent reporter assay that only detects receptors which are correctly integrated into the inner cell membrane and, thus, discriminates functional from non-functional receptor species. When we combined this method with a directed evolution procedure we obtained highly expressing mutants of the neurotensin receptor 1 with greatly improved thermostability. By this procedure receptors with poor expression and/or low stability, for which no ligands or only ones with poor binding properties are available, can now be generated in quantities allowing detailed structural and biophysical analysis.

Submission Details

ID: qQGRufTj

Submitter: Marie Ary

Submission Date: July 31, 2017, 11:46 a.m.

Version: 1

Publication Details
Klenk C;Ehrenmann J;Schütz M;Plückthun A,Sci Rep (2016) A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution. PMID:26887595
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3ZEV 2012-12-07T00:00:00+0000 3.0 Structure of Thermostable Agonist-bound Neurotensin Receptor 1 Mutant without Lysozyme Fusion
4BUO 2013-06-21T00:00:00+0000 2.75 High Resolution Structure of Thermostable Agonist-bound Neurotensin Receptor 1 Mutant without Lysozyme Fusion
4BV0 2013-06-24T00:00:00+0000 3.1 High Resolution Structure of Evolved Agonist-bound Neurotensin Receptor 1 Mutant without Lysozyme Fusion
4BWB 2013-07-01T00:00:00+0000 3.57 Structure of Evolved Agonist-bound Neurotensin Receptor 1 Mutant without Lysozyme Fusion
4GRV 2012-08-27T00:00:00+0000 2.8 The crystal structure of the neurotensin receptor NTS1 in complex with neurotensin (8-13)
4XEE 2014-12-23T00:00:00+0000 2.9 Structure of active-like neurotensin receptor
4XES 2014-12-24T00:00:00+0000 2.6 Structure of active-like neurotensin receptor
5T04 2016-08-15T00:00:00+0000 3.3 STRUCTURE OF CONSTITUTIVELY ACTIVE NEUROTENSIN RECEPTOR
6YVR 2020-04-28T00:00:00+0000 2.46 Crystal structure of the neurotensin receptor 1 in complex with the peptide full agonist NTS8-13
6Z4Q 2020-05-25T00:00:00+0000 2.92 Crystal structure of the neurotensin receptor 1 in complex with the small-molecule inverse agonist SR142948A

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.2 Neurotensin receptor type 1 variant (NTR1-D03) O88319 NTR1_MOUSE
99.8 Neurotensin receptor type 1 variant (NTR1-D03) P20789 NTR1_RAT