Structure and function in rhodopsin: Further elucidation of the role of the intradiscal cysteines, Cys-110, -185, and -187, in rhodopsin folding and function

Abstract

The disulfide bond between Cys-110 and Cys-187 in the intradiscal domain is required for correct folding in vivo and function of mammalian rhodopsin. Misfolding in rhodopsin, characterized by the loss of ability to bind 11-cis-retinal, has been shown to be caused by an intradiscal disulfide bond different from the above native disulfide bond. Further, naturally occurring single mutations of the intradiscal cysteines (C110F, C110Y, and C187Y) are associated with retinitis pigmentosa (RP). To elucidate further the role of every one of the three intradiscal cysteines, mutants containing single-cysteine replacements by alanine residues and the above three RP mutants have been studied. We find that C110A, C110F, and C110Y all form a disulfide bond between C185 and C187 and cause loss of retinal binding. C185A allows the formation of a C110-C187 disulfide bond, with wild-type-like rhodopsin phenotype. C187A forms a disulfide bond between C110 and C185 and binds retinal, and the pigment formed has markedly altered bleaching behavior. However, the opsin from the RP mutant C187Y forms no rhodopsin chromophore.Early work indicated the important role of the intradiscal domain in folding in vivo and function of rhodopsin (1). The presence of a disulfide bond between Cys-110 and Cys-187 was established (2) and it was shown later to be required for optimal signal transduction (3). A disulfide bond between two cysteines in the extracellular domain at positions equivalent to the above two cysteines in rhodopsin is known now to be conserved in most of the known G-protein-coupled receptors (4, 5). The presence of a disulfide bond indicated a tertiary structure in the intradiscal domain of rhodopsin. Strong support for this conclusion was forthcoming from extensive designed mutagenic studies as well as from studies of a large number of naturally occurring point mutations in rhodopsin that are associated with retinitis pigmentosa (RP) (6-11). Mutations in the intradiscal domain were shown to cause total or partial misfolding in vivo in rhodopsin, misfolding being operationally defined as the loss of ability to bind 11-cis retinal (6). Further, evidence was presented that misfolding results from the formation of a disulfide bond in the intradiscal domain different from the normal Cys-110-Cys-187 disulfide bond (12). Recently, Oprian and colleagues demonstrated the formation of a disulfide bond under certain conditions between Cys-185 and Cys-187 in a rhodopsin mutant reconstituted from two appropriate rhodopsin fragments (13).Further studies showed that although replacements of Cys-110 and Cys-187 by serine residues prevented folding of rhodopsin to a functional structure, corresponding replacements by alanine residues allowed the formation of the correctly folded dark-state structure. This result paralleled the earlier findings on the small-protein bovine pancreatic trypsin inhibitor, and, by analogy, suggested a globular structure for the intradiscal domain in rhodopsin (3). Recently, point mutations at the intradiscal cys...

Stability of Dark State Rhodopsin Is Mediated by a Conserved Ion Pair in Intradiscal Loop E-2

“…1B). This explanation is supported by recent findings, which show that improper disulfide bonds form as a result of mutations to this region (63)(64)(65). Furthermore, the fact that the glycosylation patterns of whole cell lysate immunoblots of mutants R177C, D190C, and D190E are different suggests the misfolding may have already occurred by the time the protein reached the endoplasmic reticulum, and thus the lack of regeneration is not simply due to an ultra-fast rate of retinal hydrolysis after regeneration (Fig.…”

G Protein-Coupled Receptor Rhodopsin: A Prospectus

“…One of the residues that exhibits the highest conservation among related proteins is Asn 55 in helix I. Its side chain is responsible for two interhelical hydrogen bonds to Asp 83 Helix II, formed by residues 71 through 100, has a length of 40 Å, is tilted ∼25° from the vector perpendicular to the plane of membranes, and is bent by 30° around tandem Gly residues, Gly 89 and Gly 90 . As a consequence of this bending, Gly 90 is in the vicinity of Glu 113 Helix III, extraordinarily important for rhodopsin, encompasses residues 106 to 139.…”

“…However, it was noted by others that Cys 185 is labeled only upon illumination (91,124), suggesting that some conformational changes are possible. This domain is further stabilized by a disulfide bond within this plug (48,90,92,125). (b) The chromophore tightens up the structure through multiple hydrophobic interactions, as well as ionic interactions between the protonated Schiff base (helix VII) and its counterion, Glu 113 (helix III).…”

Activation of G-protein-coupled receptors: a common molecular mechanism

“…In most GPCRs, mutations that disrupt this link directly affect all aspects of receptor function ( Table 1). The first evidence for the involvement of a disulfide bond in GPCR activation comes from experiments by Pederson and Ross [52] and it's role is understood best in rhodopsin and members of the rhodopsin family [44][45][46][47][48][49][53][54][55][56][57][58][59]. In all GPCRs, this disulfide linkage might be responsible for securing the interactions between TM helices and ECL2, similar to those observed in rhodopsin (Fig.…”

“…RP mutations localize to the EC, TM and cytoplasmic domains of rhodopsin and cause opsins to be either partially or completely misfolded. The misfolded fraction of the polypeptide lacks the conserved disulfide bond needed in the properly folded, functionally active fraction [47,48]. This disulfide bond is central to producing the native structure [45] and is also required for rhodopsin function [45 -49].…”