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FIG. 1. Global trace residues identify a canonical signal transduction pathway with three functional subdomains: a ligand trigger region, an allosteric linking core and a G protein-coupling region. A shows the top 20% of class A determinants (C{{alpha}} atoms) mapped onto the rhodopsin structure (1HZX [PDB] ) with retinal depicted as a yellow stick model. B shows exclusively the subset that affects ligand binding (cyan spheres) on mutation, forming the trigger region. C shows in blue the residues that cause constitutive activity or folding/expression effects on mutation. They cluster to form an intermediate linking core involved in conformational activation linking the trigger region to a coupling region shown in D (magenta spheres) consisting of residues that affect G protein coupling/activation. Ile-75 and Leu-79, which had not previously been assigned any function, are depicted as yellow spheres.
- Subtle changes were not taken into account, and given multiple functional effects a residue was classified by the one most frequently observed (Fig. 1 and Table II).
- The 39 residues ranked in the top 20th percentile (Fig. 1 and Tables I and II) are predicted to be generically important.
- To correlate the structural location of these trace residues with their function, we further sub-classified them according to their most frequently observed mutational effect and mapped this information onto the rhodopsin structure (see Fig. 1 and Supplementary Material).
- Fourteen residues, colored cyan in Fig. 1B, predominantly affect ligand binding (Table II) and segregate in the extracellular half of the cluster.
- Sixteen residues, colored blue in Fig. 1C, cause constitutive activity, folding, or expression defects, and these segregate roughly in the middle of the cluster (Table II).
- Seven residues, colored magenta in Fig. 1D, primarily affect G protein coupling and signaling, and those fill the cytoplasmic base of the transmembrane domain.
- We traced 129 rhodopsin sequences and subtracted from the resulting trace residues the global ET residues (Fig. 1) at the same percentile rank (Fig. 2).
- B shows the trace of class A sequences from Fig. 1A.
- In addition, correlation of functional effects of mutations with location revealed that the switch involves three functionally distinct but structurally connected sub-clusters: a trigger region (Fig. 1B), near the retinal binding pocket of rhodopsin, a coupling region (Fig. 1D) near the G protein-coupling site, and a linking core (Fig. 1C) between the first two.
- The seven coupling region residues fill the cytoplasmic base of the transmembrane domain, presumably forming a platform for G protein interactions, and six residues border the key G protein coupling loops 2 and 3 (Fig. 1D).
- The generic trigger region extends into the transmembrane area nearly up to the retinal binding pocket in rhodopsin (Fig. 1B), suggesting an intimate role in ligand sensing, which is supported by the mutagenesis data (Table II).
- Most trace residues, however, lie in the intermediate linking core (Fig. 1C) drawn heavily from residues forming key hydrogen bond networks such as TM1-TM2-TM7 (critical role for Asn-55), TM2-TM3-TM4 (Asn-78), and TM6-TM7 (Met-257, Asn-302, and Pro-303) (50–52).
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FIG. 2. Differential analysis identifies retinal binding site. The top 20% of trace residues (C{{alpha}} atoms) are mapped onto the rhodopsin structure (1HZX [PDB] ) with retinal depicted as yellow sticks. A shows a trace of the rhodopsin family with trace residues in orange. B shows the trace of class A sequences from Fig. 1A. C shows opsin family-specific residues obtained by subtracting B from A. They mostly cluster around the retinal binding site with a few residues found near the cytoplasmic half of the receptor. Met-207, Met-288, Phe-294, Gly-156, and Val-230 have no mutational data and are depicted as yellow spheres.
- We traced 129 rhodopsin sequences and subtracted from the resulting trace residues the global ET residues (Fig. 1) at the same percentile rank (Fig. 2).
- Most cluster around the retinal binding site (Fig. 2C), and many influence interactions with the chromophore (Table IV).
- Rhodopsin-specific residues in the cytoplasmic half of rhodopsin appear to affect G protein activation/coupling and TM-TM interactions (Fig. 2C), as revealed by mutations at Gly-51, Thr-58, and Val-250 (refer to Supplementary Material).
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FIG. 3. Constitutive activation of transducin by mutant opsins. Transducin activation by wild-type (WT) and mutant opsins is shown in the presence and absence of 11-cis-retinal and light. Transducin activity was assayed using membranes from transfected COS cells as previously described (39): {{blacktriangleup}}, in the absence of retinal; •, in the presence of retinal; and {{circ}}, time course for the reaction in the presence of retinal after exposure to light (h{{nu}}) for each mutant listed. Membrane amounts were selected to give the same light-dependent transducin activation kinetics; experiments with detergent-purified rhodopsins confirmed that the specific activities in the light were not measurably different for the mutants and wild-type (data not shown). For L79A (2X), twice the amount of membranes was assayed.
- All displayed wild-type ability to activate transducin upon exposure to light (Fig. 3).
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FIG. 4. Efficiencies of rhodopsin regeneration following photolysis. The 500-nm absorbance values of purified rhodopsins were measured in the dark. Subsequently, in the presence of a 2-fold molar excess of 11-cis-retinal, the samples were exposed to a 1-s flash of 514-nm laser light at intensity sufficient to effect complete photoisomerization. The increase in 500-nm absorbance due to regeneration was recorded over 35 min to determine the maximal percent regeneration for each protein. The values represent the averages for each mutant repeated three times, and error bars indicate the standard deviations.
- As shown in Fig. 4, substitutions at both positions (W175A, W175C, W175H, and L79A) led to constitutive activity, i.e.
- To determine the ability of the mutants to regenerate the dark photoreceptive state following photoactivation, we used a laser flash to effect nearly instantaneous quantitative photoisomerization of bound 11-cis-retinal to the all-trans form in the presence of excess 11-cis-retinal and then measured the maximum extent of regeneration by absorbance at 500 nm (Fig. 4).