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. 2012 Jun 19;109(25):9810-5.
doi: 10.1073/pnas.1202107109. Epub 2012 Jun 4.

Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations

Karola M Schlinkmann  1 , Annemarie Honegger, Esin Türeci, Keith E Robison, Daša Lipovšek, Andreas Plückthun
Affiliations

Critical features for biosynthesis, stability, and functionality of a G protein-coupled receptor uncovered by all-versus-all mutations

Karola M Schlinkmann et al. Proc Natl Acad Sci U S A. .

Abstract

The structural features determining efficient biosynthesis, stability in the membrane and, after solubilization, in detergents are not well understood for integral membrane proteins such as G protein-coupled receptors (GPCRs). Starting from the rat neurotensin receptor 1, a class A GPCR, we generated a separate library comprising all 64 codons for each amino acid position. By combining a previously developed FACS-based selection system for functional expression [Sarkar C, et al. (2009) Proc Natl Acad Sci USA 105:14808-14813] with ultradeep 454 sequencing, we determined the amino acid preference in every position and identified several positions in the natural sequence that restrict functional expression. A strong accumulation of shifts, i.e., a residue preference different from wild type, is detected for helix 1, suggesting a key role in receptor biosynthesis. Furthermore, under selective pressure we observe a shift of the most conserved residues of the N-terminal helices. This unique data set allows us to compare the in vitro evolution of a GPCR to the natural evolution of the GPCR family and to observe how selective pressure shapes the sequence space covered by functional molecules. Under the applied selective pressure, several positions shift away from the wild-type sequence, and these improve the biophysical properties. We discuss possible structural reasons for conserved and shifted residues.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Sequence variability of rNTR1-D03 (A) and GPCR class A consensus (C). Positions in a snake plot (Left) and homology model of rNTR1-D03 (Right) are color coded in a gradient from red for high mutational tolerance to blue for low tolerance (see SI Text for details). Letters indicate conserved sequence motifs in each helix. Positions that were not randomized (flexible N terminus and C terminus) or for which no 454 data were obtained (13 transmembrane positions) are coded in gray. (B) Histograms of observed amino acid frequencies (red) compared with the amino acid frequency distribution expected for an unbiased library (blue) illustrate the significance of the different rmsd levels.
Fig. 2.
Fig. 2.
Correlation between sequence constraints in natural GPCRs and in the rNTR1-D03 454 sequencing experiment. Colors indicate whether the amino acid distribution in a given position is equally constrained in both systems (purple), more constrained in natural class A GPCR sequences (blue), or in the deep sequencing from rNTR1-D03 (red). The size of the circles indicates the constraints imposed by the respective system; large circles indicate positions with high constraints.
Fig. 3.
Fig. 3.
Sequence shifts. Positions are colored according to conservation (blue) or shift (red) away from the rNTR1-D03 sequence. Uppercase letters refer to robust effects and lowercase letters to effects obscured by codon bias in the raw amino acid consensus. The strength of the conservation or shift is given by the rmsd of the amino acid distribution from the input distribution as shown in Fig. 1A and listed in Fig. S7. For the highlighted positions, the influence of mutations on the performance of the receptor has been further characterized (Table 1).
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