Molecular basis for variations in the sensitivity of pathogenic rhodopsin variants to 9-cis-retinal.

Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. Of these apparent class II variants, 67 exhibit a measurable increase in expression in the presence of 9-cis-retinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts relative to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest that many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (ΔN73, S131P, and R135G) and show that two of these retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.

Disease-linked supertrafficking of a potassium channel.

Gain-of-function (GOF) mutations in the voltage-gated potassium channel subfamily Q member 1 (KCNQ1) can induce cardiac arrhythmia. In this study, it was tested whether any of the known human GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface-"supertrafficking." Seven of the 15 GOF mutants tested were seen to surface traffic more efficiently than the WT channel. Among these, we found that the levels of R231C KCNQ1 in the plasma membrane were fivefold higher than the WT channel. This was shown to arise from the combined effects of enhanced efficiency of translocon-mediated membrane integration of the S4 voltage-sensor helix and from enhanced post-translational folding/trafficking related to the energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with the voltage-gated potassium channel-regulatory subfamily E member 1 not only exhibited constitutive conductance but also revealed that the single-channel activity of this mutant is only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the effects of supertrafficking and constitutive channel activation, which together offset reduced channel activity. These investigations show that membrane protein supertrafficking can contribute to human disease.

Cotranslational folding stimulates programmed ribosomal frameshifting in the alphavirus structural polyprotein.

Viruses maximize their genetic coding capacity through a variety of biochemical mechanisms, including programmed ribosomal frameshifting (PRF), which facilitates the production of multiple proteins from a single mRNA transcript. PRF is typically stimulated by structural elements within the mRNA that generate mechanical tension between the transcript and ribosome. However, in this work, we show that the forces generated by the cotranslational folding of the nascent polypeptide chain can also enhance PRF. Using an array of biochemical, cellular, and computational techniques, we first demonstrate that the Sindbis virus structural polyprotein forms two competing topological isomers during its biosynthesis at the ribosome-translocon complex. We then show that the formation of one of these topological isomers is linked to PRF. Coarse-grained molecular dynamics simulations reveal that the translocon-mediated membrane integration of a transmembrane domain upstream from the ribosomal slip site generates a force on the nascent polypeptide chain that scales with observed frameshifting. Together, our results indicate that cotranslational folding of this viral protein generates a tension that stimulates PRF. To our knowledge, this constitutes the first example in which the conformational state of the nascent polypeptide chain has been linked to PRF. These findings raise the possibility that, in addition to RNA-mediated translational recoding, a variety of cotranslational folding or binding events may also stimulate PRF.

Divergent Folding-Mediated Epistasis Among Unstable Membrane Protein Variants.

Many membrane proteins are prone to misfolding, which compromises their functional expression at the plasma membrane. This is particularly true for the mammalian gonadotropin-releasing hormone receptor GPCRs (GnRHR). We recently demonstrated that evolutionary GnRHR modifications appear to have coincided with adaptive changes in cotranslational folding efficiency. Though protein stability is known to shape evolution, it is unclear how cotranslational folding constraints modulate the synergistic, epistatic interactions between mutations. We therefore compared the pairwise interactions formed by mutations that disrupt the membrane topology (V276T) or tertiary structure (W107A) of GnRHR. Using deep mutational scanning, we evaluated how the plasma membrane expression of these variants is modified by hundreds of secondary mutations. An analysis of 251 mutants in three genetic backgrounds reveals that V276T and W107A form distinct epistatic interactions that depend on both the severity and the mechanism of destabilization. V276T forms predominantly negative epistatic interactions with destabilizing mutations in soluble loops. In contrast, W107A forms positive interactions with mutations in both loops and transmembrane domains that reflect the diminishing impacts of the destabilizing mutations in variants that are already unstable. These findings reveal how epistasis is remodeled by conformational defects in membrane proteins and in unstable proteins more generally.

The Effects of Codon Usage on Protein Structure and Folding.

The rate of protein synthesis is slower than many folding reactions and varies depending on the synonymous codons encoding the protein sequence. Synonymous codon substitutions thus have the potential to regulate cotranslational protein folding mechanisms, and a growing number of proteins have been identified with folding mechanisms sensitive to codon usage. Typically, these proteins have complex folding pathways and kinetically stable native structures. Kinetically stable proteins may fold only once over their lifetime, and thus, codon-mediated regulation of the pioneer round of protein folding can have a lasting impact. Supporting an important role for codon usage in folding, conserved patterns of codon usage appear in homologous gene families, hinting at selection. Despite these exciting developments, there remains few experimental methods capable of quantifying translation elongation rates and cotranslational folding mechanisms in the cell, which challenges the development of a predictive understanding of how biology uses codons to regulate protein folding. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

General trends in the effects of VX-661 and VX-445 on the plasma membrane expression of clinical CFTR variants.

Cystic fibrosis (CF) is caused by mutations that compromise the expression and/or function of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. Most people with CF harbor a common misfolded variant (ΔF508) that can be partially rescued by therapeutic "correctors" that restore its expression. Nevertheless, many other CF variants are insensitive to correctors. Using deep mutational scanning, we quantitatively compare the effects of two correctors on the plasma membrane expression of 129 CF variants. Though structural calculations suggest corrector binding provides similar stabilization to most variants, it's those with intermediate expression and mutations near corrector binding pockets that exhibit the greatest response. Deviations in sensitivity appear to depend on the degree of variant destabilization and the timing of misassembly. Combining correctors appears to rescue more variants by doubling the binding energy and stabilizing distinct cotranslational folding transitions. These results provide an overview of rare CF variant expression and establish new tools for precision pharmacology.

Molecular basis for the evolved instability of a human G-protein coupled receptor.

Membrane proteins are prone to misfolding and degradation. This is particularly true for mammalian forms of the gonadotropin-releasing hormone receptor (GnRHR). Although they function at the plasma membrane, mammalian GnRHRs accumulate within the secretory pathway. Their apparent instability is believed to have evolved through selection for attenuated GnRHR activity. Nevertheless, the molecular basis of this adaptation remains unclear. We show that adaptation coincides with a C-terminal truncation that compromises the translocon-mediated membrane integration of its seventh transmembrane domain (TM7). We also identify a series of polar residues in mammalian GnRHRs that compromise the membrane integration of TM2 and TM6. Reverting a lipid-exposed polar residue in TM6 to an ancestral hydrophobic residue restores expression with no impact on function. Evolutionary trends suggest variations in the polarity of this residue track with reproductive phenotypes. Our findings suggest that the marginal energetics of cotranslational folding can be exploited to tune membrane protein fitness.