Systematic profiling of temperature- and retinal-sensitive rhodopsin variants by deep mutational scanning

Abstract

Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9- cis -retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9- cis -retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.

Molecular Basis for Variations in the Sensitivity of Pathogenic Rhodopsin Variants to 9-cis-Retinal

“…Nevertheless, relatively few variants exhibit gains in expression that approach the upper bound (Figure 4A), which suggests the response to retinal is also likely sensitive to other effects of these mutations. ( 20 )…”

“…The proteostatic effects of retinal varies considerably across the spectrum of destabilized rhodopsin variants. ( 16, 18, 20 ) We therefore repeated these experiments in the presence of 5 μM 9- cis -retinal to identify retinopathy variants that are most amenable to correction. Consistent with our recent findings,( 20 ) the results show that poorly expressed variants exhibit the largest change in expression in the presence of retinal (Figure 3A).…”

“…( 16, 18, 20 ) We therefore repeated these experiments in the presence of 5 μM 9- cis -retinal to identify retinopathy variants that are most amenable to correction. Consistent with our recent findings,( 20 ) the results show that poorly expressed variants exhibit the largest change in expression in the presence of retinal (Figure 3A). There are clusters of highly responsive variants that include several previously uncharacterized mutations near the binding pocket (L47R and T289P) and the cytosolic interface (L57R and ΔN73) (Table S1, Figure 3B).…”

Chromenone derivatives as novel pharmacological chaperones for retinitis pigmentosa-linked rod opsin mutants

“…To learn if CR4 and CR5 compounds could promote the correct transport of other disease-related rod opsin mutants, we used a recently reported deep mutational scanning assay to survey their effects on the plasma membrane expression of 123 rod opsin variants related to RP (29)(30)(31). Of note, a few CSNB variants were included in this screening.…”

“…Moreover, these compounds showed a stabilizing effect for the pathogenic P23H rod opsin during biosynthesis resulting in the improved transport of this variant to the cell surface. Furthermore, we surveyed the potential of these new chromenone derivatives to rescue membrane targeting of other misfolding Rho variants by using a recently described deep mutational scanning approach, identifying a panel of mutants most amenable to be rescued by one of these molecules (29)(30)(31).…”