From Evolutionary trace of G protein-coupled receptors reveals clusters of residues that determine global and class-specific functions.
The Journal of biological chemistry.
The Journal of biological chemistry.
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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).