Photoactivation Perturbs the Membrane-embedded Contacts between Sensory Rhodopsin II and Its Transducer

Abstract

The photoactivation mechanism of the sensory rhodopsin II (SRII)-HtrII receptor-transducer complex of Natronomonas pharaonis was investigated by time-resolved Fourier transform infrared difference spectroscopy to identify structural changes associated with early events in the signal relay mechanism from the receptor to the transducer. Several prominent bands in the wild-type SRII-HtrII spectra are affected by amino acid substitutions at the receptor Sensory rhodopsin II (SRII)1 functions as a phototaxis receptor for blue light avoidance in several halophilic archaea (1-4). This protein belongs to a growing family of microbial rhodopsins, seven-helix retinylidene membrane proteins now found in all domains of life (5-8). The SRII receptor forms a tight intermolecular complex with its cognate transducer protein HtrII in the cell membrane. The transducer, like its homologous methyl-accepting chemotaxis transducers, possesses two transmembrane helices and a large cytoplasmic domain that binds at its distal end a His-kinase that phosphorylates a flagellar motor switch regulator (1, 9).Interactions of HtrII with the SRII receptor are localized to the transducer transmembrane and membrane-proximal domains (10, 11). A crystal structure of SRII bound to an Nterminal HtrII fragment containing the transmembrane helices (TM1 and TM2) shows tight van der Waals interaction and three hydrogen bonds between TM2 and SRII helices F and G (12). An atomic structure of the membrane-proximal domain of the transducer is not available; however, fluorescent probe accessibility and Förster resonance energy transfer measurements show interaction of this domain with the cytoplasmic E-F loop of the receptor (13).The signal relay mechanism from SRII to HtrII in the complex has become a focus of interest in the past several years, in part because of its importance to the general understanding of interaction between integral membrane proteins. In accord with the unified model for transport and signaling by microbial rhodopsins (14, 15), the key event in the transducer activation is an outward tilt of the receptor helix F during the lifetime of its M intermediate, which has been directly detected by EPR of paramagnetic probes in the free receptor (16) and in its complex with transducer (17). In a response to the helix F movements, the TM2 of HtrII is displaced from its initial position (17). Additional structural changes were observed in the cytoplasmic membrane proximal region by fluorescent probe accessibility measurements (13).Fourier transform infrared (FTIR) difference spectroscopy has been used extensively in the past to elucidate structural changes in the photocycles of several microbial rhodopsins (18 -27). This technique measures changes in the infrared absorption of protein groups and enables studies of light-induced conformational changes without the necessity of introducing potentially structure-perturbing probes. Because of its sensitivity to small changes in hydrogen bonding, it is especially well suited for the study of interacti...

Early Photocycle Structural Changes in a Bacteriorhodopsin Mutant Engineered to Transmit Photosensory Signals

“…The BR-T protein offers a way to help distinguish functionally important versus relatively unimportant light-induced changes Therefore the structural changes of the HOOP vibrations, the O-H stretching vibration of the introduced Thr residue (A215T), and a distorted ␣-helix appear to be important for signal generation. The results from several different methods show that light-induced structural changes occur all along the SRII⅐HtrII interface, which includes the region on the periplasmic side of the membrane (41), the membrane-embedded domain (42)(43)(44), and the cytoplasmic membrane-proximal domain (45,46). The structural changes detected in this study further suggest that the signal relay occurs within the membrane-embedded contact surfaces.…”

“…The next steps in signal relay and signal propagation through HtrII require further study. According to data from several laboratories (3), these structural changes may induce outward F-helix tilting of SRII (48), structural changes of binding surfaces between SRII and HtrII (16,43,44), rotation of TM2 of HtrII, and structural changes in the membrane proximal domain (HAMP) of HtrII (45,49) to relay the signal to the phosphorylation cascade controlling the flagellar motor switch.…”

Three strategically placed hydrogen-bonding residues convert a proton pump into a sensory receptor

“…Evidence for such coupling is that the nearby Tyr-199-Asn-74 hydrogen bond (Fig. 1b) is greatly perturbed, probably disrupted, in the signaling state (20). Our results argue for crucial signal relay chemistry occurring within the membrane ''BR'' designates BR in the following description: 100-ms 500-nm stimulus for SRII͞HtrII (a), 500-ms 580-nm stimulus for P200T͞V210Y-BR (b), 500-ms 550-nm stimulus for A215T-BR (c), P200T͞V210Y͞A215T-BR͞NpHtrII (d), and P200T͞ V210Y͞A215T-BR͞G83F-NpHtrII (j), and 100-ms 550-nm stimulus for P200T͞ V210Y͞A215T-BR͞HsHtrII (e).…”

“…The results from several different methods show that light-induced structural changes occur all along the SRII-HtrII interface (refs. 17,(20)(21)(22) V. Bergo, E. Spudich, K. Rothschild, and J.L.S., unpublished data). However, which, if any, of these changes are essential and whether they are involved in the signal relay process within the complex or in later steps in signal propagation are unknown (23).…”

Mining Protein Interaction Groups

“…The X-ray structure of 1h2s was obtained at 1.93 Å resolution (Gordeliy et al, 2002) and it provided an atomic picture of the first step of the signal transduction. The interactions in the sRII-HtrII complex have been intensively investigated to find the signal relay mechanism from the receptor to the transducer (Bergo et al, 2005;Inoue et al, 2007;Sudo et al, 2007). The 3D structure of the interactions is shown in Fig.…”