Light-induced structural changes occur in the transmembrane helices of the Natronobacterium pharaonis HtrII transducer.
The Natronobacterium pharaonis HtrII (NpHtrII) transducer interacts with its cognate photoactive sensory rhodopsin receptor, NpSRII, to mediate phototaxis responses. NpHtrII is predicted to have two transmembrane helices and a large cytoplasmic domain and to form a homodimer. Single cysteines were substituted into an engineered cysteine-less NpHtrII at 38 positions in its transmembrane domain. Oxidative disulfide cross-linking efficiencies of the monocysteine mutants were measured with or without photoactivation of NpSRII. The rapid cross-linking rates at several positions support that NpHtrII is a dimer when functionally expressed in the Halobacterium salinarum membrane. Thirteen positions in the second transmembrane segment (TM2) exhibited significant light-induced increases in cross-linking efficiency, and they define a single face traversing the length of the segment when modeled as an alpha-helix. Four positions in this helix showing light-induced decreases in efficiency are clustered on the cytoplasmic side of the protein. One of the monocysteine mutants, G83C, showed loss of phototaxis responses, and analysis of double mutants showed that the G83C mutation alters the dark structure of the TM2-TM2' region of NpHtrII. In summary, the results reveal conformationally active regions in the second transmembrane segment of NpHtrII and a face along the length of TM2 that becomes more available for TM2-TM2' cross-linking upon receptor photoactivation. The data also establish that one residue in TM2, Gly83, is critical for maintaining the proper conformation of NpHtrII for signal relay from the photoactivated receptor to the kinase-binding region of the transducer.
Yang
C S
CS
Department of Microbiology and Molecular Genetics, University of Texas Medical School at Houston, 6431 Fannin Street, Houston, Texas 77030, USA.
Spudich
J L
JL
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects*; D002338:Carotenoids / Q000378:metabolism; D016764:Cell Polarity; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D019617:Halobacterium salinarum / Q000235:genetics; D025602:Halorhodopsins; D055537:Light Signal Transduction; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019633:Natronobacterium / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000528:radiation effects; D025603:Sensory Rhodopsins
D019843:Archaeal Proteins; D004220:Disulfides; D025602:Halorhodopsins; C434451:HtrII protein, Natronobacterium pharaonis; D008565:Membrane Proteins; D011994:Recombinant Proteins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D003545:Cysteine