Abstract
The G protein-coupled receptor opsin is a phospholipid scramblase that facilitates rapid transbilayer phospholipid exchange in liposomes. The mechanism by which opsin scrambles lipids is unknown. It has been proposed that lipid translocation may occur at protein-protein interfaces of opsin dimers. To test this possibility, we rationally engineered QUAD opsin by tryptophan substitution of four lipid-facing residues in transmembrane helix 4 (TM4) that is known to be important for dimerization. Atomistic molecular dynamics simulations of wild type and QUAD opsins combined with continuum modeling revealed that the tryptophan substitutions lower the energetically unfavorable residual hydrophobic mismatch between TM4 and the membrane, reducing the drive of QUAD opsin to dimerize. We purified thermostable wild type and QUAD opsins, with or without a SNAP tag for fluorescence labeling. Single molecule fluorescence measurements of purified SNAP-tagged constructs revealed that both proteins are monomers. Fluorescence-based activity assays indicated that QUAD opsin is a fully functional scramblase. However, unlike wild type opsin which dimerizes en route to insertion into phospholipid vesicles, QUAD opsin reconstitutes as a monomer. We conclude that an engineered opsin monomer can scramble phospholipids, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange.
Highlights
Phospholipids flip-flop rapidly across disc membranes of retinal rod photoreceptor cells in an ATP-independent manner[1,2]
Molecular dynamics simulations combined with continuum modeling of the energetics of protein-lipid interactions indicated that the effect of the tryptophan substitutions is to lower the energetically costly residual hydrophobic mismatch between transmembrane helix 4 (TM4) and the membrane, and to reduce the drive of quadruple mutant opsin (QUAD) opsin to dimerize via the TM4 interface
To design an opsin construct with impaired dimerization, we had the choice of modifying transmembrane (TM) helix 1 (TM1) and/or TM4, as both these helices have been strongly implicated in G protein-coupled receptor (GPCR) dimerization[9]
Summary
Kalpana Pandey[1], Birgit Ploier[1], Michael A. To address the role of opsin dimerization in lipid scrambling, we initially tested the scramblase activity of previously reported opsin mutants bearing amino acid substitutions in transmembrane (TM) helices 1 and 5 that have been proposed to be important for dimerization of opsin as well other GPCRs9–12 To this end, we used a reconstitution-based approach that was designed to reveal the effect of a particular mutation on opsin’s scramblase activity, and to indicate with considerable precision the oligomeric state of the protein as it inserts into preformed vesicles during detergent-mediated reconstitution[13,14]. Using this approach we found that certain rhodopsin point mutants in TM1 and TM5 that are associated with autosomal dominant retinitis pigmentosa, reconstitute into vesicles as monomers but retain wild-type (WT)-like scramblase activity[13] These studies indicated that opsin dimerization is not required for lipid scrambling and suggested a novel disease mechanism based on dimerization deficiency[13]. We conclude that an engineered opsin monomer can scramble phospholipids, i.e. a dimer interface is not required for scrambling, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange
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