Abstract
The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Forster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Forster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.
Highlights
Archaeal sensory rhodopsins I and II (SRI and sensory rhodopsin II (SRII))1 are membrane-embedded phototaxis receptors that modulate the motility apparatus of Halobacterium salinarum and related haloarchaeal species [1,2,3]
Light Decreases the Accessibility of E-F Loop Position 154 in the SRII-HtrII Complex, but Not in Free SRII—As a first method to examine the effect of association with HtrII and of photoactivation on the SRII cytoplasmic loop structure, we measured fluorescent probe accessibilities of Cys residues introduced at 8 cytoplasmic surface residues (1 on the A-B, 1 on the C-D, and 6 on the E-F loop) of SRII
The accessibilities in the dark of the residues on the A-B and C-D loops were unaffected by the presence of HtrII, whereas HtrII reduced the accessibility at 3 positions on the E-F loop (151, 154, and 158), position 154 most strongly (Fig. 1)
Summary
Archaeal sensory rhodopsins I and II (SRI and SRII) are membrane-embedded phototaxis receptors that modulate the motility apparatus of Halobacterium salinarum and related haloarchaeal species [1,2,3]. Light-induced tilting of helix F (and to a lesser extent helix G) opens a cytoplasmic channel in BR [14] and this tilting has been shown to occur in SRII both when free and bound to HtrII [15, 16], with HtrII inhibiting the proton uptake through the channel as in the SRI-HtrI complex This conformational change, which involves structural alterations mainly in the cytoplasmic end of helix F and in the E-F loop [14], has been proposed based on several lines of evidence to be responsible for activating the Htr transducers [17]
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