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

Abstract

Two forms of the phototaxis receptor sensory rhodopsin I distinguished by differences in its photoactive site have been shown to be directly correlated with attractant and repellent signaling by the dual-signaling protein. In prior studies, differences in the photoactive site defined the two forms, namely the direction of light-induced proton transfer from the chromophore and the pK a of an Asp counterion to the protonated chromophore. Here, we show by both in vivo and in vitro measurements that the two forms are distinct protein conformers with structural similarities to two conformers seen in the light-driven proton transport cycle of the related protein bacteriorhodopsin. Measurements of spontaneous cell motility reversal frequencies, an in vivo measure of histidine kinase activity in the phototaxis system, indicate that the two forms are a photointerconvertible pair, with one conformer activating and the other inhibiting the kinase. Protein conformational changes in these photoconversions monitored by site-directed spin labeling show that opposite structural changes in helix F, distant from the photoactive site, correspond to the opposite phototaxis signals. The results provide the first direct evidence that displacements of helix F are directly correlated with signaling and impact our understanding of the sensory rhodopsin I signaling mechanism and the evolution of diverse functionality in this protein family.

Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin

“…SRI and SRII have been shown to undergo a similar movement of helix F in their light-induced conversions between E and C conformers. 26,27 A two-conformer model of BR is supported well by the library of structures obtained from structures of spectral intermediates in the BR photocycle. 39 The structures of the dark-state conformation and the early intermediates K and L are approximated well by the E conformer, while the structures of late intermediates M–O are approximated well by the C conformer.…”

“…36 Light-induced displacement/rotation of helix F in the photocycle of the SRI–HtrI attractant signaling is in the direction that is the opposite of that in BR and SRII, which agrees with other data showing that SRI undergoes a C → E conversion in the SRI–HtrI molecular complex. 24,26,37 …”

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

“…Projection maps obtained by cryo-electron microscopy suggested in addition a photoinduced movement of helix 7 (110). The outward movement of helix 6 (accompanied in BR by more subtle rearrangements of the cytoplasmic portions of helices 3, 5, and 7) is the major conformational change that occurs during the M1→M2 transition in BR (111), NaR (38), SRI (112) and SRII (112–113). However, structural rearrangement of helix 2 appears to be unique for CCRs and is thought to play a major role in formation of a conducting pore (99).…”

Mechanism divergence in microbial rhodopsins

“…The isolated SRII protein in the dark is in the E conformation, as shown by (i) its near superimposable helix positions to the BR E conformer [23], (ii) its light-induced Schiff base proton release outward to the aspartate residue corresponding to Asp85 in BR [24–25], (iii) its light-induced E → C transition according to helix F motion assessed by EPR [26–27], (iv) the similarity of late photocycle backbone changes of BR and SRII measured by FTIR [28], and (v) its ability to pump protons when free of its transducer HtrII, as first found for transducer-free SRI [29–30] showing that these sensory rhodopsins must switch Schiff base connectivity during the conformational change [6, 9]. In both SRI and SRII, the binding of their cognate Htr transducers block their proton pumping activity [31–32].…”