Divergent Epistatic Interactions Arise from Distinct Folding Defects in an Unstable Membrane Protein

Core Concepts

Mutations that disrupt cotranslational membrane integration (V276T) or tertiary structure (W107A) of the gonadotropin-releasing hormone receptor (GnRHR) form distinct epistatic interactions that depend on the mechanism and severity of destabilization.

Abstract

The authors investigated how epistatic interactions between mutations are shaped by the folding defects in an unstable membrane protein, the gonadotropin-releasing hormone receptor (GnRHR). They compared the epistatic interactions formed by two GnRHR variants - V276T, which disrupts cotranslational membrane integration, and W107A, which disrupts the native tertiary structure.
Key insights:

Mutations that form negative epistatic interactions with V276T are predominantly located in soluble loops, while mutations that form positive epistatic interactions with W107A are found across both loops and transmembrane domains.
The distinct epistatic patterns arise from differences in how the V276T and W107A mutations destabilize the protein. V276T, which disrupts cotranslational folding, exhibits more pronounced negative epistasis, while W107A, which disrupts the native fold, exhibits positive epistasis as additional destabilizing mutations have diminishing impacts.
An unsupervised clustering analysis revealed that mutations in transmembrane domains tend to form positive epistatic interactions with W107A, likely because the protein is already predominantly misfolded in this background.
The findings suggest that the distinct biosynthetic mechanisms of membrane proteins can differentially shape their fitness landscapes through context-dependent epistatic interactions.

Stats

Mutations that disrupt cotranslational membrane integration (V276T) reduce the plasma membrane expression of GnRHR by 65 ± 4% relative to wild-type.
Mutations that disrupt the native tertiary structure (W107A) reduce the plasma membrane expression of GnRHR by 88 ± 4% relative to wild-type.

Quotes

"Mutations that generate Stage I and Stage II folding defects form distinct epistatic interactions throughout this receptor."
"An unsupervised learning analysis reveals how these interactions depend on both changes in stability and the topological context of the mutation."

Key Insights Distilled From

Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants

by Chamness,L. ... at www.biorxiv.org 08-27-2023

https://www.biorxiv.org/content/10.1101/2023.08.25.554866v3

Deeper Inquiries

How do the distinct epistatic interactions observed for V276T and W107A GnRHR variants translate to differences in their evolutionary trajectories?

The distinct epistatic interactions observed for the V276T and W107A GnRHR variants have implications for their evolutionary trajectories. The negative epistatic interactions seen with V276T suggest that mutations that compromise the cotranslational membrane integration of GnRHR tend to synergistically decrease its expression. This indicates that the destabilizing effects of these mutations are compounded when combined, leading to a more significant reduction in protein expression. In contrast, the positive epistatic interactions observed with W107A suggest that mutations that destabilize the native tertiary structure of GnRHR have a less pronounced effect on protein expression when combined with W107A. This is likely due to the already marginal surface expression of W107A, where even highly destabilizing mutations can only cause a relatively small decrease in the yield of folded protein.
In terms of evolutionary trajectories, these findings suggest that the evolutionary paths of V276T and W107A variants may be influenced by the types of mutations they interact with. The negative epistasis observed with V276T may constrain the mutational landscape, as mutations that further compromise cotranslational folding efficiency are likely to be selected against due to their additive destabilizing effects. On the other hand, the positive epistasis seen with W107A may allow for a broader range of mutations to be tolerated, as the destabilizing effects of these mutations are attenuated in the context of an already unstable protein. This could potentially lead to a more diverse set of evolutionary outcomes for the W107A variant compared to the V276T variant.

How generalizable are these findings to other classes of membrane proteins, and what insights could they provide into the evolution of membrane protein families?

While the study focused on the specific case of GnRHR variants, the findings are likely to be generalizable to other classes of membrane proteins. Membrane proteins share common features in terms of their cotranslational folding and stability constraints, which are governed by the unique environment of the lipid bilayer. Therefore, the mechanisms of epistatic interactions observed in the context of unstable membrane protein variants, as demonstrated with V276T and W107A, are likely to apply to other membrane proteins as well.
These findings provide insights into the evolution of membrane protein families by highlighting the role of stability-mediated epistasis in shaping the mutational landscape of these proteins. The differential effects of destabilizing mutations on protein expression and folding efficiency, as seen with V276T and W107A, can influence the evolutionary trajectories of membrane proteins by determining the mutational tolerance and the range of permissible genetic interactions. Understanding how destabilizing mutations interact with different regions of membrane proteins and how these interactions impact protein stability and folding can shed light on the evolutionary constraints and adaptive potential of membrane protein families. By studying the epistatic interactions in membrane proteins, we can gain a better understanding of how these proteins evolve and adapt to changing environmental conditions.