Hydrogen bonding alteration of Thr-204 in the complex between pharaonis phoborhodopsin and its transducer protein.
Pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronobacterium pharaonis. It forms a 2:2 complex with its transducer protein, pHtrII, in membranes and transmits light signals through the change in the protein-protein interaction. We previously found that the ppR(K) minus ppR spectrum in D(2)O possesses vibrational bands of ppR at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII [Furutani, Y., Sudo, Y., Kamo, N., and Kandori, H. (2003) Biochemistry 42, 4837-4842]. A D/H-unexchangeable X-H group appears to form a stronger hydrogen bond upon retinal photoisomerization in the ppR-pHtrII complex. This article aims to identify the group by use of various mutant proteins. According to the crystal structure, Tyr-199 of ppR forms a hydrogen bond with Asn-74 of pHtrII in the complex. Nevertheless, the 3479 (-)/3369 (+) cm(-1) bands were preserved in the Y199F mutant, excluding the possibility that the bands are O-H stretches of Tyr-199. On the other hand, Thr-204 and Tyr-174 form a hydrogen bond between the retinal chromophore pocket and the binding surface of the ppR-pHtrII complex. These FTIR measurements revealed that the bands at 3479 (-)/3369 (+) cm(-1) disappeared in the T204A mutant, while being shifted to 3498 (-) and 3474 (+) cm(-1) in the T204S mutant. They appear at 3430 (-)/3402 (+) cm(-1) in the Y174F mutant. From these results, we concluded that the bands at 3479 (-)/3369 (+) cm(-1) originate from the O-H stretch of Thr-204. A stronger hydrogen bond as shown by a large spectral downshift (110 cm(-1)) suggests that the specific hydrogen bonding alteration of Thr-204 takes place upon retinal photoisomerization, which does not occur in the absence of the transducer protein. Thr-204 has been known as an important residue for color tuning and photocycle kinetics in ppR. The results presented here point to an additional important role of Thr-204 in ppR for the interaction with pHtrII. Specific interaction in the complex that involves Thr-204 presumably affects the decay kinetics and binding affinity in the M intermediate.
Sudo
Yuki
Y
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Furutani
Yuji
Y
Shimono
Kazumi
K
Kamo
Naoki
N
Kandori
Hideki
H
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D002338:Carotenoids / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D017666:Deuterium Oxide / Q000737:chemistry; D025602:Halorhodopsins; D006860:Hydrogen Bonding; D019633:Natronobacterium / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010649:Phenylalanine / Q000235:genetics; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics; D025603:Sensory Rhodopsins; D012694:Serine / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D013912:Threonine / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D019843:Archaeal Proteins; D025602:Halorhodopsins; D020283:Photoreceptors, Microbial; D025603:Sensory Rhodopsins; C434450:phototaxis receptor sensory rhodopsin II, Natronobacterium pharaonis; C061508:sensory rhodopsin II protein, archaeal; D013912:Threonine; D002338:Carotenoids; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D017666:Deuterium Oxide; D000409:Alanine