Constitutive activity in chimeras and deletions localize sensory rhodopsin II/HtrII signal relay to the membrane‐inserted domain

Abstract

SummaryHalobacterium salinarum sensory rhodopsin II (HsSRII) is a phototaxis receptor for blue-light avoidance that relays signals to its tightly bound transducer HsHtrII (H. salinarum haloarchaeal transducer for SRII). We found that disruption of the salt bridge between the protonated Schiff base of the receptor's retinylidene chromophore and its counterion Asp73 by residue substitutions D73A, N or Q constitutively activates HsSRII, whereas the corresponding Asp75 counterion substitutions do not constitutively activate Natronomonas pharaonis SRII (NpSRII) when complexed with N. pharaonis haloarchaeal transducer for SRII (NpHtrII). However, NpSRII D75Q in complex with HsHtrII is fully constitutively active, showing that transducer sensitivity to the receptor signal contributes to the phenotype. The swimming behaviour of cells expressing chimeras exchanging portions of the two homologous transducers localizes their differing sensitivities to the HtrII transmembrane domains. Furthermore, deletion constructs show that the known contact region in the cytoplasmic domain of the NpSRII-NpHtrII complex is not required for phototaxis, excluding the domain as a site for signal transmission. These results distinguish between the prevailing models for SRII-HtrII signal relay, strongly supporting the 'steric triggertransmembrane relay model', which proposes that retinal isomerization directly signals HtrII through the mid-membrane SRII-HtrII interface, and refuting alternative models that propose signal relay in the cytoplasmic membrane-proximal domain.

Opposite Displacement of Helix F in Attractant and Repellent Signaling by Sensory Rhodopsin-Htr Complexes

“…One way to describe the photointerconvertibility of the two conformers of SRI-HtrI is that each conformer acts as a photoreceptor mediating in one case an attractant and the other case a repellent response, and each conformer is a constitutively activated form of the other. The constitutively activated mutant SRII D73N was identified and shown to elevate cell reversal frequencies by 3.1-fold over cells expressing wild-type SRII (29,36). We tested here another mutation at the same position in SRII D73Q -HtrII, and we observes a 10.2-fold increase in spontaneous reversal frequency in the dark over the wild-type value (Fig.…”

“…At the low natural wild-type SR-Htr complex concentrations, the adaptation reactions reset the kinase activity, and consequently the swimming reversal frequency, to its prestimulus value (35). However, when the receptor-transducer complexes are overexpressed in the cells to Ͼ10-fold levels, the adaptation system is not sufficient to reset the kinase activation level (29,36). This effect allows us to use the spontaneous reversal frequency to compare the kinase modulation of different complexes under constant conditions, specifically darkness or continuous light.…”

“…Wild-type SRII under continuous saturating 500-nm illumination produces the same reversal frequency as SRII D73Q in the dark, indicating SRII D73Q appears to be fully constitutively activated. Yet illumination of the SRII D73Q -HtrII complex does not produce an attractant response (36), and in fact, the mutant complex exhibits the same elevated reversal frequency in the light as in the dark (Fig. 7).…”

Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping

“…It has been shown by solid state NMR spectroscopy, fluorescence and EPR spectroscopy that the E-F loop is interacting tightly with the transducer [38][39][40] . However, it is clear that the E-F loop is not crucial for signal transduction, as NpSRII E-F loop deletion mutants as well as BR triple mutant, which has a small disordered E-F loop, are all capable of signaling 20,41 . The remaining question is whether the E-F loop enhances the signal transduction from NpSRII to NpHtrII or just serves for NpSRII stabilization.…”

Salinibacter Sensory Rhodopsin

“…HsSRI and NpSRII form 2:2 signaling complexes with their cognate halobacterial transducer proteins (Htr), HsHtrI and NpHtrII, in membranes, and transmit light signals through changes in protein-protein interactions (4). The excitation light absorbed by HsSRI and NpSRII triggers trans-cis isomerization of the retinal chromophore that is covalently bound to a conserved lysine residue via a protonated Schiff base linkage (5,6).…”