Divergent Pairwise Epistasis in the Context of Unstable Membrane Protein Variants

Many eukaryotic membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for mammalian gonadotropin-releasing hormone receptors (GnRHRs), which are G protein-coupled receptors involved in reproductive steroidogenesis. We recently demonstrated that evolutionary modifications within mammalian GnRHRs appear to have coincided with adaptive changes in cotranslational folding efficiency. Though changes in protein stability are known to shape evolutionary interactions, it is unclear how the energetic drivers of cotranslational folding in the membrane may modify epistatic interactions. We therefore surveyed the pairwise epistatic interactions that modify the expression of two destabilized GnRHR variants bearing mutations that selectively compromise either its membrane topology (V276T) or its native tertiary structure (W107A). Using deep mutational scanning (DMS), we evaluated how the effects of these mutations on the expression of the mature form of the protein at the plasma membrane are modified by hundreds of secondary mutations. A focused analysis of 251 mutants with high-quality measurements in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the degree to which they destabilize the protein and the mechanism of their destabilization. An unsupervised learning analysis shows that V276T forms predominantly negative epistatic interactions that are most pronounced among destabilizing mutations within soluble loop regions. In contrast, W107A forms interactions with mutations in both loops and transmembrane domains that skew positive as a result of the diminishing impact of the destabilizing mutations in the context of an already unstable variant. These findings provide general insights into how pairwise epistasis is remodeled by conformational defects in membrane proteins and, more generally, in unstable proteins.