Mechanism of transient dark activity of 13-desmethylretinal/rod opsin complex [4]

Full text of DOI:10.1021/ja9827752

J. Am. Chem. Soc. 1998, 120, 12357-12358
12357
Mechanism of Transient Dark Activity of
13-Desmethylretinal/Rod Opsin Complex
Qiang Tan and Koji Nakanishi*
Department of Chemistry
Columbia UniVersity
New York, New York 10027
Rosalie K. Crouch
Figure 1. Traces A-H: Relative activities of incubation mixture
measured by transducin assay. A. 13dm 1 and opsin; B. All-trans-retinal
and opsin; C. All-trans-retinal and opsin pretreated with â-ionone; D.
2H13dm 2 and opsin; E. Ret6 3 and opsin; F. 13dm 1 and opsin pretreated
with â-ionone; G. â-ionone and opsin; H. Opsin. Trace I: A497 of 13dm
1 with opsin. Trace J: A497 of 13dm 1 and opsin pretreated with â-ionone.
Department of Ophthalmology
Medical UniVersity of South Carolina
Charleston, South Carolina 29425
ReceiVed August 4, 1998
We report the studies employing 11-cis-13-desmethylretinal
(13dm) 1, its dihydro analogue 2H13dm 2, and 11-cis six-
membered ring-locked analogue ret6 3, to clarify the mechanism
of dark activity of the 13-desmethylretinal/rod opsin complex.
Rhodopsin (Rh) is composed of a retinal chromophore linked
to Lys 296 of the apoprotein opsin via a protonated Schiff base
(PSB).1 Light-induced retinal 11-cis f trans isomerization
triggers protein conformational changes leading to metarhodopsin
II (Meta-II) which activates the G-protein transducin to initiate
the enzymatic cascade of visual transduction.2 Phosphorylation
of Meta-II by rhodopsin kinase and subsequent binding of arrestin
deactivate Meta-II.3
process of visual regulation. Following the 1980 report that 13dm
1 and opsin induced dark activation of phosphodiesterase,7 a
subsequent assay for rhodopsin kinase activity showed that the
dark activity decayed with time, i.e., it is transient.4c In the
following, the mechanism of this transient dark activity has been
studied with analogues 1-38a by performing UV/Vis/CD mea-
surements and transducin assays,8b in which the relative activity
of opsin/retinal complex is measured by counting the amount of
GTPγ35S bound to transducin.
Figure 1 shows the transducin assay and UV/Vis measurements.
Opsin treated with 13dm 1 exhibited transient dark activity (trace
A), consistent with previous kinase assay data.4c Decay of the
activity was accompanied with the regeneration of 13-desmethyl
rhodopsin (13dmRh) with a 497 nm λmax (trace I), implying that
an early active species is replaced by the inactive 13dmRh with
the PSB linkage (497 nm). The residual activity after 120 min
incubation could be due to the nonspecific binding of GTPγ35S.
Recently it has been observed that opsin can be activated by
retinal analogues including all-trans-retinal, all-trans-13dm 4, all-
trans-C15 5, and â-ionone 6 in the absence of light.4 Dark activity
The retinal binding cavity contains a binding site (site I in
Figure 3 below) for the cyclohexene ring of â-ionone 6,4d,9 which
activates opsin, but compared to 13dm 1, only weakly.4c-e Traces
J and F show that â-ionone inhibits the binding and opsin
activation of 13dm 1. Hence, for opsin activation, 13dm 1 needs
to enter the protein cavity with the cyclohexene ring occupying
the corresponding binding pocket. In contrast, traces B and C
show all-trans-retinal exhibited sustained activity which was
essentially unaffected by â-ionone, in agreement with the
published data.4d 2H13dm 210 (prepared as in Scheme 1) and
of opsin/retinal complex has been proposed to be responsible for
bleaching adaptation,4c-d,5 whereas constitutive activation of opsin
leads to certain night blindness and retinal degeneration diseases.5d,6
Therefore, clarification of the mechanism of dark activity of opsin/
retinal complex is essential for understanding the complicated
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(8) (a) 13dm 1 was prepared according to: Broek, A. D.; Muradin-
Szweykowska, M.; Courtin, J. M. L.; Lugtenburg, J. J. R. Neth. Chem. Soc.
1983, 102, 46-51. (b) To 10 mM pH 7.4 Tris containing 4 µM opsin or
opsin pretreated with 20 equiv of â-ionone for 1 h was added 4 molar equiv
retinal analogues in DMF (<2% v/v). According to the published procedures
(Wessling-Resnick, M.; Johnson, G. L. J. Biol. Chem. 1987, 262, 12444-
12447), a portion of the above mixture was transferred into the transducin
assay solution in 10 mM pH 7.4 Tris, which then contained 1-10 µM
transducin, 200 nM opsin, and 10 µM GTPγ35S. After 5 min 70 µL of the
assay mixture was filtered through a nitrocellulose filter, which was dissolved
in 10 mL of scintillation fluid Filtron-X, and its radioactivity, corresponding
to the relative activity of opsin/retinal complex, was counted on a Beckman
scintillation counter. CD and UV/Vis spectra were taken in 10 mM pH 7.4
Tris and 23 mM dodecyl maltoside.
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10.1021/ja9827752 CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/13/1998

12358 J. Am. Chem. Soc., Vol. 120, No. 47, 1998
Communications to the Editor
Cotton effect around 280 nm that decays with loss in activity. In
contrast, opsin incubated with ret6 3 (Figure 2B) showed neither
activity (Figure 1, trace E) nor a negative 280 nm CD Cotton
effect (Figure 2B). It should be noted that in the case of native
Rh, the physiologically active Meta-II has a negative 280 nm CD
band as opposed to the positive 280 nm CD of Rh.12 Hence, it
is possible that the transient dark active species of opsin
incorporating 13dm 1 or 2H13dm 2 adopts a Meta-II-like
conformation.
On the basis of these data, we propose the following rational-
ization for the transient dark actiVation of opsin by 13dm 1 and
2H13dm 2 (Figure 3; the 3D structure is based on photoaffinity
cross-linking1f). The opsin cavity incorporating the retinals is
divided into three binding domains, site I for the cyclohexene
ring, site II for the polyene chain, and site III for PSB.
(i) Occupation of site I by the cyclohexene moiety is required
for opsin activation.
(ii) Prior to pigment formation, 11-cis-retinals transiently
activate the protein during the binding process. Rhodopsin kinase
assay study4c showed that 13dm 1 exhibits no transient dark
activation when Lys 296 is dimethylated. Thus, approach of the
retinal aldehyde close to Lys 296 in site III or formation of a
Schiff base linkage is necessary for opsin activation.
Figure 2. Time-dependent CD spectra of opsin incubated with retinal
analogues. (A) 13dm 1 (0 °C). (B) Ret6 3 (rt). (C) 2H13dm 2 (rt).
(iii) Protonation of the Schiff base (Figure 3A) inactivates the
protein through salt bridge formation between PSB and its
counteranion Glu 113.6a,13
(iv) During the process of “anchoring” of retinal in sites I (as
in i) and III (as in ii), the polyene chain adopts a shape which
gives rise to a transient Meta-II-like conformation in opsin.
In the case of 13dm 1 and 2H13dm 2, as the chromophore
enters the opsin binding site with its cyclohexene ring in site I
and the unprotonated Schiff base linkage in site III, the retinal
polyene enters site II and adjusts the torsions around C10-C13
to attain the conformation responsible for protein activation
(Figure 3B). The ring-locked ret6 3 cannot adopt this conforma-
tion and is thus inactive (Figure 3C). No dark activity has been
reported for 11-cis-retinal;4c a possible explanation is that since
the binding of 11-cis-retinal is 10 times faster than 13dm 1, the
retinal and protein rapidly attain the inactive state of rhodopsin.
Experiments designed to slow this binding process may reveal
transient activation. Note that traces B and C in Figure 1, which
denote sustained dark activity by all-trans-retinal, are different
from the present transient dark activity. It is regarded that in
the case of sustained activity, the chromophore lies outside the
binding site;4d however, further studies are necessary.
Figure 3. Proposed model of activity of opsin/13dm 1 complex. 3D
model shows seven R-helices surrounding the retinal binding cavity
including sites I-III. (A) The activation is transient since PSB formation
at site III leads to inactivation of the protein. (B) Prior to PSB formation,
as the retinal cyclohexene ring occupies site I and the aldehyde approaches
Lys 296 at site III without PSB formation, 13dm analogues 1 and 2 are
able to adjust the polyene conformation at site II to transiently adopt a
shape close to Meta-II rhodopsin. (C) Ret6 3 is inactive since the ring-
fixed polyene, unlike the 13dm analogues, cannot achieve the Meta-II-
like conformation.
Scheme 1a
Finally, the opsin used for the experiments was from a rod out
segment preparation14 and contains mainly the rod opsin. In
isolated salamander rods, a similar response is noted, i.e.,
activation without light by â-ionone 64a and 13dm 1.4b However,
in isolated salamander cones, the opposite result is obtained as
â-ionone deactivated the basal activity of the photoreceptor, acting
as an inverse agonist.5c Therefore, the interaction of the opsin
with the ligand appears to be dependent on the structure of the
opsin and may explain some of the differences in the properties
of rods and cones.
a Conditions: (a) DIBAL-H, CH2Cl2, -78 °C, 78%; (b) diethyl (1-
cyanoethyl)phosphonate, NaH, THF, 76% overall; (c) DIBAL-H, CH2Cl2,
-78 °C, 80%; (d) â-ionyltriphenylphosphonium bromide, BuLi, THF,
65%; (e) amberlyst acidic resin, acetone, 95%; (f) diethyl cyanomethyl
phosphonate, NaH, THF, quant.; (g) DIBAL-H, CH2Cl2, -78 °C, silica
gel chromatography to separate 1:1 isomers, 79% overall.
ret6 311 formed pigments with opsin, with λmax at 270 and 508
nm, respectively. As shown in Figure 1, 2 exhibited transient
activity similar to 13dm 1 (traces A, D), while 3 was inactive
(trace E).
Acknowledgment. We thank Drs. Babak Borhan and Jihong Lou for
discussions, Dr. Craig A. Parish for assistance with the transducin assay
setup, and NIH Grants GM36564 and EY04939 and the Foundation to
Prevent Blindness for funding.
The conformational changes of retinal analogues and opsin
during activation were studied by CD (Figure 2). For 13dm 1
and 2H13dm 2 which display transient activity (Figure 1, traces
A, D), the CD shown in Figure 2A,C both exhibit a negative
JA9827752
(11) Prepared according to: de Grip, W. J.; van Oostrum, J.; Bovee-Geurts,
P. H. M.; van der Steen, R.; van Amsterdam, L. J. P.; Groesbeek, M.;
Lugtenburg, J. Eur. J. Biochem. 1990, 191, 211-220.
(10) 1H NMR (CDCl3, 400 MHz, ppm) for 2: δ 9.54 (1H, d, J ) 7.9 Hz),
6.89 (1H, dt, J1 ) 15.6 Hz, J2 ) 12.9 Hz), 6.18 (1H, ddt, J1 ) 15.6 Hz, J2 )
7.9 Hz, J3 ) 1.4 Hz), 6.07 (1H, d, J ) 16.6 Hz), 6.01 (1H, d, J ) 16.2 Hz),
5.40 (1H, t, J ) 6.6 Hz), 2.47 (2H, m), 2.42 (2H, m), 2.02 (2H, t, J ) 6.3
Hz), 1.83 (3H, s), 1.71 (3H, s), 1.64 (2H, m), 1.50 (2H, m), 1.04 (6H, s). nOe
was observed between 9-Me/7-H, 9-Me/11-H, and 8-H/10-H. HRMS: Cal-
culated 272.2140 for C19H28O, observed 272.2137.
(12) Okada, T.; Matsuda, T.; Kandori, H.; Fukada, Y.; Yoshizawa, T.;
Shichida, Y. Biochemistry 1994, 33, 4940-4946.
(13) (a) Cohen, G. B.; Oprian, D. D.; Robinson, P. R. Biochemistry 1992,
31, 12592-12601. (b) Sakmar, T. P.; Franke, R. R.; Khorana, H. G. Proc.
Natl. Acad. Sci. U.S.A. 1989, 86, 8309-8313.
(14) Opsin prepared according to: Papermaster, D. S. Methods Enzymol.
1982, 81, 49-52.