Constitutive activation of opsin: Influence of charge at position 134 and size at position 296

Abstract

In previous studies, mutation of Lys296 or Glu113 in opsin has been shown to result in constitutive activation of the protein--that is, these mutants can activate the G protein transducin in the absence of chromophore and in the absence of light. These and other data have led to the suggestion that a salt bridge between Lys296 and Glu113 helps to constrain opsin to an inactive conformation. It is shown here that of 12 different amino acids substituted at position 296, all, except Arg and the wild-type Lys, are constitutively active at neutral pH, lending further support to this suggestion. However, activation of opsin appears also to be influenced significantly by the size of amino acid side chain at position 296. Thus, there are multiple effects of the mutations. Wild-type opsin is also shown to be weakly active at pH 6.1. Five other charged amino acids in the membrane-embedded region of the protein (Asp83, Glu122, Glu134, Arg135, and Glu201) were mutated to see if they affect constitutive activity. Of these amino acids, only mutation of Glu134 results in an increase in the activity of opsin. Changing Glu134 to Gln increases the activity of opsin, while changing Glu134 to Asp inhibits activity. These results suggest that a negative charge on Glu134 is important in stabilizing the inactive state of opsin. Glu134 is highly conserved in all visual pigments and most of the other G protein-linked receptors.

Crystal structure of the ligand-free G-protein-coupled receptor opsin

“…In agreement with this observation, the E134Q opsin mutant showed increased activity towards the G protein, suggesting that removal of the negative charge at Glu-134, e.g. by protonation, facilitates formation of the active receptor conformation [91,92,135]. In rhodopsin, the energy which is provided by photon absorption and used for retinal cis/trans isomerization shifts the pK a for proton uptake and formation of the Meta II conformation to higher values compared with formation of the opsin* conformation [136,137].…”

“…The role of the active cytoplasmic conformation of Meta II in catalyzing nucleotide exchange in the Gα subunit was investigated by peptide competition experiments [124], mutagenesis [94,126,135,140,141,142], and biochemical assays [143]. These studies showed that some key residues (e.g.…”

“…These studies showed that some key residues (e.g. Glu Previous mutagenesis, fluorescence and EPR studies [71,72,73,86,135,144] suggested that the disruption of the "ionic lock" as observed in the opsin* structure is induced by a rotational and translational movement of TM6. The opsin* crystal structure revealed that due to TM6…”

“…Cohen and co-workers reported that at pH 6.1 wild-type opsin displayed, when compared with Meta II, a small activity towards transducin [135]. Mutagenesis studies of two residues (Glu-113 and Lys-296) showed a strongly increased, pH-dependent constitutive activity of opsin when one interaction partner of the Lys-296/Glu-113 salt bridge was removed (Table 4.1) [92,93,135,159,160].…”

“…Mutagenesis studies of two residues (Glu-113 and Lys-296) showed a strongly increased, pH-dependent constitutive activity of opsin when one interaction partner of the Lys-296/Glu-113 salt bridge was removed (Table 4.1) [92,93,135,159,160]. In contrast to the E113Q or K296G mutants, the K296R opsin mutant was inactive like wild-type opsin at pH 6.7 [92,93,135]. Therefore disruption of the salt bridge between Lys-296 (or Arg-296 in the K296R mutant) and the counterion Glu-113 appears to be decisive for forming the active conformation.…”