title: Functional importance of the interhelical hydrogen bond between Thr204 and Tyr174 of sensory rhodopsin II and its alteration during the signaling process.
abstract: Sensory rhodopsin II (SRII), a receptor for negative phototaxis in haloarchaea, transmits light signals through changes in protein-protein interaction with its transducer HtrII. Light-induced structural changes throughout the SRII-HtrII interface, which spans the periplasmic region, membrane-embedded domains, and cytoplasmic domains near the membrane, have been identified by several studies. Here we demonstrate by site-specific mutagenesis and analysis of phototaxis behavior that two residues in SRII near the membrane-embedded interface (Tyr174 on helix F and Thr204 on helix G) are essential for signaling by the SRII-HtrII complex. These residues, which are the first in SRII shown to be required for phototaxis function, provide biological significance to the previous observation that the hydrogen bond between them is strengthened upon the formation of the earliest SRII photointermediate (SRII(K)) only when SRII is complexed with HtrII. Here we report frequency changes of the S-H stretch of a cysteine substituted for SRII Thr204 in the signaling state intermediates of the SRII photocycle, as well as an influence of HtrII on the hydrogen bond strength, supporting a direct role of the hydrogen bond in SRII-HtrII signal relay chemistry. Our results suggest that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174, which is located at both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface to the signal-bearing second transmembrane helix of HtrII. The results argue for a critical process in signal relay occurring at this membrane interfacial region of the complex.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas 77030, USA.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Sudo
authors.forename: Yuji
authors.initials: Y
authors.lastname: Furutani
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori
authors.forename: John L
authors.initials: JL
authors.lastname: Spudich
mesh_terms: D002462:Cell Membrane; D019617:Halobacterium salinarum / Q000737:chemistry / Q000235:genetics; D025602:Halorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D017550:Spectroscopy, Fourier Transform Infrared; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D025602:Halorhodopsins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D013912:Threonine; D014443:Tyrosine