TY - JOUR
T1 - Spectroscopic study of the transmembrane domain of a rhodopsin–phosphodiesterase fusion protein from a unicellular eukaryote
AU - Watari, Masahito
AU - Ikuta, Tatsuya
AU - Yamada, Daichi
AU - Shihoya, Wataru
AU - Yoshida, Kazuho
AU - Tsunoda, Satoshi P.
AU - Nureki, Osamu
AU - Kandori, Hideki
N1 - Funding Information:
This work was supported by Japanese Ministry of Education, Culture, Sports, Science, and Technology Grants 17J30010 and 18K14634 (to W. S.), 18K06109 (to S. P. T.) and 16H06294 (to O. N.), and 25104009, 15H02391, and 18H03986 (to H. K.); by CREST, Japan Science and Tech-nology Agency, Grant JPMJCR1753 (to H. K.); and by PRESTO, Japan Science and Technology Agency, Grant JPMJPR1688 (to S. P. T.). The authors declare that they have no conflicts of interest with the contents of this article.
Publisher Copyright:
© 2019 Watari et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/3/8
Y1 - 2019/3/8
N2 - The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an “M” intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the “K” intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the -helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.
AB - The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an “M” intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the “K” intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the -helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.
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U2 - 10.1074/jbc.RA118.006277
DO - 10.1074/jbc.RA118.006277
M3 - Article
C2 - 30622140
AN - SCOPUS:85062605570
VL - 294
SP - 3432
EP - 3443
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 10
ER -