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Structure

Article

The Signaling Pathway of Rhodopsin
Yifei Kong1 and Martin Karplus1,2,*
1
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA
2
Laboratoire de Chimie Biophysique, ISIS, Université Louis Pasteur, 67000 Strasbourg, France
*Correspondence: marci@tammy.harvard.edu
DOI 10.1016/j.str.2007.04.002

SUMMARY more than 50 years (Hubbard and Wald, 1952; Wald,
1968), the mechanism by which this occurs is still obscure.
The signal-transduction mechanism of rho- Rhodopsin consists of the protein opsin, which is com-
dopsin was studied by molecular dynamics posed of seven transmembrane helices (helices I–VII),
(MD) simulations of the high-resolution, inactive a short additional helix (helix VIII) approximately parallel
structure in an explicit membrane environment. to the membrane, and a set of connecting loops on the
The simulations were employed to calculate two sides of the membrane, plus the 11-cis retinylidene
chromophore bound covalently to Lys296 through a pro-
equal-time correlations of the fluctuating inter-
tonated Schiff-base linkage (Okada et al., 2004; Palczew-
action energy of residue pairs. The resulting
ski et al., 2000; Teller et al., 2001). The chromophore is
interaction-correlation matrix was used to de- buried in the middle of the transmembrane helical bundle,
termine a network that couples retinal to the far from the rhodopsin cytoplasmic surface, which is the
cytoplasmic interface, where transducin binds. binding interface between rhodopsin and the G protein.
Two highly conserved motifs, D(E)RY and The isomerization of retinal takes place on the 200 fs
NPxxY, were found to have strong interaction timescale (Schenkl et al., 2005), while metarhodopsin II
correlation with retinal. MD simulations with re- (meta II), the active species, is formed on the millisecond
straints on each transmembrane helix indicated timescale through a complex cycle comprised of a series
that the major signal-transduction pathway in- of intermediates including bathorhodopsin, lumirhodop-
volves the interdigitating side chains of helices sin, and metarhodopsin I (meta I), which are formed after
retinal isomerization on a nanosecond, microsecond,
VI and VII. The functional roles of specific resi-
and millisecond timescale, respectively (Menon et al.,
dues were elucidated by the calculated effect
2001; Okada et al., 2001). They have been identified pri-
of retinal isomerization from 11-cis to all-trans marily through spectral shifts of the primary retinal absorp-
on the residue-residue interaction pattern. It is tion band (Okada et al., 2001). The overall structures of
suggested that Glu134 may act as a ‘‘signal bathorhodopsin (ns) and lumirhodopsin (ms) appear to be
amplifier’’ and that Asp83 may introduce a very similar to the inactive protein, as shown by recent
threshold to prevent background noise from X-ray crystallography (Nakamichi and Okada, 2006a,
activating rhodopsin. 2006b). Meta I keeps essentially the same helical posi-
tions, an orientation illustrated by cryo-electron micros-
copy (Ruprecht et al., 2004). Only when the system
reaches meta II, which is required for normal activation
INTRODUCTION of the G protein, transducin, do larger structural changes
appear (Nakamichi and Okada, 2006b). The states have
Rhodopsin is a membrane protein that detects light in the also been studied by a variety of other methods, including
rod photoreceptor cell. Like other G protein-coupled re- Cys scanning mutagenesis and site-directed spin labeling
ceptors (GPCRs), rhodopsin exists in equilibrium between (for a review, see Hubbell et al. [2003]). There is indirect ev-
its activated and inactivated forms in vivo. This equilibrium idence, based primarily on spin-label mobility changes,
is controlled by the isomerization of retinal, the cofactor that there are significant displacements of certain helices
covalently bound to rhodopsin. In the dark state, retinal (particularly helix VI) at their cytoplasmic ends. The pro-
is in its 11-cis form and stabilizes rhodopsin in its inactive posed ‘‘outward’’ motion of helix VI was confirmed by dis-
conformation. In the presence of light, retinal is photoiso- tance change estimates from spin-label interactions and
merized to the all-trans form, which activates rhodopsin. disulfide crosslinking (Fritze et al., 2003). Although it has
Activated rhodopsin catalyzes the replacement of GDP been suggested that helix VI is particularly flexible due
by GTP on the a subunit of a heterotrimetric G protein, to looser packing, there is no evidence for this from the
transducin, which is bound to rhodopsin. This, in turn, trig- X-ray B factors of the inactive structure. Some of the
gers the response of the rod cells in the retina (Filipek changes observed in photoactivation are also observed
et al., 2003; Gether and Kobilka, 1998; Meng and Bourne, by corresponding techniques in constitutively active mu-
2001). Although the activation of rhodopsin by retinal tants of rhodopsin (Kim et al., 1997). How the outward
isomerization from 11-cis to all-trans has been known for motion of helix VI and the smaller motions of other helices

Structure 15, 611–623, May 2007 ª2007 Elsevier Ltd All rights reserved 611

The equilibrium constant mean-square deviations (rmsds) of Ca atoms (Figure S1) is very far on the side of the former to avoid false signals. The fluctuations of chromophore to the cytoplasmic regions requires that the interaction energies are obtained from ensembles of there be long-range communication within the protein. simulations of rhodopsin in the inactive (11-cis retinal) structure and the changes in the interaction energies Residue-Based Interaction-Energy Correlation that occur on a short timescale (i. a higher-resolution structure of none. matrix.. 2004. Wyman.. 1993). totaling 16 ns) on that structure a method based on equal-time correlations of the interac. The charged residues. 2002. Structure Signaling Pathway of Rhodopsin lead to activation. 2004. 2004) was released. 2003. interestingly... is not clear. became available (Okada et al. the NPxxY motif (residues 302–306) in helix VII. identified two highly conserved sequence motifs that have After high-resolution structures of inactive rhodopsin been shown to play critical roles (Stenkamp et al. Lemaitre retinal and its isomerization. With the isomer. retinal isomerization on the interaction network.. are involved in acti. However. Teller et al. Finally. 1997. 2005). see sets of residue pairs in equilibrium molecular dynamics Supplemental Data 11. We rationale for the present study is similar to that used in the begin with the former and discuss the later in the following conservation correlation analysis based on multiple GPCR subsections.. presumably through the cytoplasmic sequences (Lockless and Ranganathan.e. we find that. the simulations permit us to determine ducin. we are able to identify a network that extends from Alternatively.. Although tural alterations. While the current simula- seconds. see Supplemental Data 1 conformation requires a time course on the order of milli.. in a monomer like the GPCR rhodopsin. and the other one is and normal-mode studies were performed (Crozier et al. This indicates that analysis of the MD tion to the all-trans form has taken place. signal shows that. 611–623. 2005).. May 2007 ª2007 Elsevier Ltd All rights reserved . we have performed simulations (four the source of the tertiary allosteric coupling. Although the MWC model was proposed for multisubunit RESULTS proteins. 2004.. dynamics of inactive rhodopsin. For details. The results indicate that the interaction tion-energy fluctuations (i. The pres- 2003. studies (Lemaitre et al. Huber et al. only ter- tiary structural changes. loops of rhodopsin in contact with the a subunit of trans.. Overall Simulation Characteristics vation. 1999. an analysis of the role of the helices in transmitting the nism is involved. with same setup. 2001). Kim et al. Weitz and Nathans. and the residue-based root-mean-square fluctuation although constitutively active rhodopsins exist (Fritze (rmsf) (Figure S2) are comparable to those of previous et al. Palczewski et al. and Changeux (MWC) in their land- mark paper published 40 years ago (Brunori et al.. An essential question is how the isom. the correlation between two correlation pattern is not sensitive to the choice of sets of interaction energies between pairs of amino acids initial crystal structure (PDB ID 1HZX or 1U19) or to the at a given time.e.. a series of molecular-simulation the cytoplasmic terminus of helix III. we calculated the 612 Structure 15. For the trajectories. 2002). in accord with the model proposed sential role. thus. 2004. 2002). it is the fluctuations of rium between the inactive and active forms (Changeux the side chains rather than main chains that play the es- and Edelstein. 2007. By use of the interaction-energy correlation librium in the inactive. for certain et al. it is possible to examine the changes in the the retinal-binding pocket to the cytoplasmic surface. 2007.. 2005). of the protein is the objective of this paper.. high-resolution structure (2. while the change to the active second timescale.’’ but involves a propagation of more localized struc. It is generally presumed that an allosteric-type mecha. to the cytoplasmic Comparisons of amino acid sequences for GPCRs have surface. Rohrig et al. rhodopsin. Grossfield ent simulations show how these two motifs are coupled to et al. Huber et al. the energetic origins of the coupling. To determine Saam et al. at least on the nano- ization occurring in 200 fs. 2003. in which there is a pre-existing equilib.. For details. 2006. The signal transduction provided by rhodopsin from the erization of retinal to the all-trans form. averaged over the simulation) between all presence of internal water molecules. it is very likely that the transition is not ‘‘all or tions were in progress. which are not avail- erization signal propagates efficiently from the middle of able from the conservation analysis. This suggests that inactive performed with a total simulation length of 22 ns (see rhodopsin (with 11-cis retinal) and meta II (also with Experimental Procedures). the flexibility and dynamics of the protein Eight independent MD simulations of rhodopsin were must play the essential roles.’’ The investigation into how retinal isomerization ID 1HZX)... 2005) and experimental B factors The equilibrium constant shifts to meta II when isomeriza. One of these is the D(E)RY motif (residues 134–136) at 2000. Suel et al. available with this article online.. 2001). 2007. The correlation induced by chromophore isomerization. (Teller et al. Saam et al. 10 ns) after the isom. in part to permit comparison with the work of provides a signal that propagates to the cytoplasmic end others (Crozier et al. by definition.. where retinal is located. though there are simulations can provide a meaningful description of the no details on the relative concentrations. Isin et al. rhodopsin conformers generated by molecular dynamics One approach is to investigate the correlation of the dy- (MD) simulation of the inactive rhodopsin structure in namic behavior between different structural units at equi- a membrane. the root- 11-cis retinal) are in equilibrium. 2003).. 2005. Also.. there is a strong anisotropic effect of results are described briefly in the Discussion. by Monod. This can be termed ‘‘tertiary allosteric we completed our analysis with the older structure (PDB coupling.6 Å). we use equal-length trajectories. rhodopsin (Okada et al.. Faraldo-Gomez et al.. To initiate the analysis.

(C) The interaction energy between residues 135–249 and 94–296 shows a strong negative correlation (0.038). with the same colors used in this panel. blue. action-energy correlation between four residue pairs gies of any two residue pairs throughout the molecule. Val13) (Figure 1). Structure 15. (B) The interaction energy between residues 139–248 and 135–249 shows a strong positive correlation (0. we of time in the 22 ns MD runs and then used the results to show the residue pair interaction-energy profile and inter- determine equal-time correlations of the interaction ener. Thr94/Lys249. (Arg135/Glu249. The interaction-energy profiles between pairs of interactions during the 22 ns simulation are shown in (B)–(D). 611–623. interaction energies between all residue pairs as a function To illustrate the interaction correlation analysis.626). (D) The interaction energy between residues 135–139 and 135–249 shows little correlation (0. Interaction Correlation (A) Four pairs of residue-residue interactions are shown in an equilibrated rhodopsin structure (gray). see Supplemental Data 2. red. The Ca atoms of each residue are rendered as spheres. May 2007 ª2007 Elsevier Ltd All rights reserved 613 . 94–296. A ‘‘control’’ simulation on the same set of mental analyses that have focused on certain helices. two residue pairs in the absence of the protein showed we then determined which of the secondary structural that the long-range interaction-energy correlation be- elements play an important role in signal transduction by tween 94/296 and 135/249 is essentially zero.Structure Signaling Pathway of Rhodopsin Figure 1. and Arg135/ Because of the helical structure of rhodopsin and experi. and orange. Val139/Lys245. restraining them. The interactions between residues 139–248. and 135–139 are illustrated by double-headed arrows in green.673). For details. respectively. 135–249.

..825 non-zero data points in (see Supplemental Data 4).. the former is normalized between 0 and 1.e. due to local resulting matrix is shown in Figure 2A.104 and a standard deviation of 0. On the extracellular side. residues. and the positions of the seven transmembrane helices are shown at the bottom. 190. To eliminate background noise. Schiff base. the show relatively strong coupling with retinal. the column/row index is the sequence index of rhodopsin from 1 to 348. Yan et al. 2003). is likely to first change the residue-inter.206 (see Supplemen. is the essential quantity for analysis. also see Supplemen- duction. the loop (190–201) tion in rhodopsin. On the cytoplasmic side. All of the residues in the retinal-binding whether it is direct or is transmitted through other residue pocket that are highlighted in Figure 3A are also perturbed interactions).’’ including retinal) and tal Data 3). the origin of the signal (‘‘Lys296. Structure Signaling Pathway of Rhodopsin Figure 2. The usual correlations (i. Figure 3A shows to obtain a residue-based correlation matrix (see Experi. 208.. 94 113. In both matrices. the initial perturbation. the residue correlation matrix. 2004. The residue corre. The arrow indicates the position (residue 296) to which retinal is bound. In the residue correlation matrix. (B) Residue-based displacement correlation of rhodopsin. 122. To obtain information about the signal-transduction a condensed correlation matrix (993 3 993) was built by mechanism. May 2007 ª2007 Elsevier Ltd All rights reserved . The 1026 3 1026 interac. 611–623. tal Data 5. The distribution of the absolute values Correlation of Retinal Interactions: of the correlations of the pairwise interaction energies is Signal-Transduction Pathway shown in Figure S3A. Interaction-Energy Correlation Matrix and Residue For comparison.e. Many residues (90. Residue 181 is also correlated with residue 296. action. such as retinal isomeriza. identified two groups of residues with strong coupling to 614 Structure 15. Both quantities are normalized. close residues (see Experimental Procedures). fore. 1984) of the 22 ns MD sim- interaction energies larger than 1 kcal/mol in magnitude ulation trajectories (see Experimental Procedures) was were identified among the 348 residues of rhodopsin constructed. a color-coded scheme of the magnitude of the interaction mental Procedures). There. correlations of the type illustrated in Figures 1A and 1B. we structure. which gives the correlations between correlation. The resulting interaction matrix was projected the rest of rhodopsin on the structure. which are symmetric. by retinal isomerization (see below). but there is no evidence for long-range correlations data. Glu113 forms a salt bridge to the protonated dues in the system. There are a total of 525. in particular. although there is no direct inter- eventually reach the rhodopsin cytoplasmic side. between the b sheet and helix V shows relatively strong action pattern around the retinal-binding pocket and correlation with retinal. the residue-residue displacement Correlation Matrix of Rhodopsin crosscorrelation matrix (Figure 2B) based on a quasi- A total of 1026 residue-residue interactions with average harmonic analysis (Levy et al. and the later is normalized between 1 and +1. independent of how it arises (i. 181. which represents the which is consistent with experimental observations (Patel interaction coupling between any residue pairs in the et al. and 298) in the retinal-binding pocket (within 5 Å) and row represents a specific residue in the protein. lation matrix shows the coupling between any two resi. Comparison of Interaction Correlation with Displacement Correlation from Quasi-Harmonic Analysis (A) Residue correlation matrix of rhodopsin (348 3 348). each column 297. this matrix with an average value of 0. we map the correlated interactions between introducing a correlation cutoff of 0.. In the process of intramolecule signal trans. and residues within a helix have large correlations) are tion-energy correlation matrix was constructed from these seen.102.

i. 94. show in Figure 5B only the perturbations of interactions dent 1 ns and one 9 ns MD simulation of all-trans retinal with an average interaction energy greater than 5 kcal/ were performed (see Experimental Procedures). 122. structure obtained by averaging the eight simulations. 113. see Equation 5 in Exper- are given in Supplemental Data 7. which Structure 15. see Supplemental Data 9. Most pair interactions close the side chains of the two helices. relative to meta I (Fritze et al. 207.. Of these. Not surprisingly. and 139). 1997. weaker. the net- served in hemoglobin simulations (Gelin et al. includes Asp83 on helix II. 292. residues on he. 289. it is useful to focus on specific inter- restraining other helices. 2005. 91.. Glu134.. and 308 (Figure 3A). Fritze et al. 2006b). 116. which is located around the NPxxY the double-bond cis/trans isomerization (Nakamichi and motif. The main effect is that the retinal molecule be- lular loop. 208. 1997. actions rather than the residues. it is im. and 298.e. We found experimental observation that 9-demethyl-retinal has a continuous coupling pathway from retinal to the NPxxY only weak activation properties and with its photo-isomer- motif through helix VII. terestingly. 95. are Given the helix-bundle structure of rhodopsin. Densely clustered groups of changes by Retinal Isomerization in the interactions are observed around both the D(E)RY To complement the interaction coupling analysis based on motif and the NPxxY motif (Cai et al.. The overall structure does network are anisotropic. and the torsion engendered by the isom- around the D(E)RY motif. Results of retinal isomerization. This obser- all-trans retinal. the interaction partners. shown in Figure 4B. however. The mol in absolute value. and 294. 292. Network: Importance of Charged Residues ing that they are essential for the signal transduction from To analyze the changes in the interaction network due to retinal to the rhodopsin cytoplasmic interface. the residue coupling 212. retinal. VII (303–308) (Figure 3H) were found to significantly weaken the coupling between retinal and the rhodopsin Effects of Retinal Isomerization on the Interaction cytoplasmic interface. Arg147 on the second intracel. vation suggests a mechanism for conduction of a signal. as mimicked in the simulation Arg314 (see Figure 5B) are important (see also below). 301. 188... 208. 114. Based on only 14 interactions (86. 2005). work of changes spreads from retinal to more distant re- gions. toplasmic loops. the changes in the interaction coupling tails. May 2007 ª2007 Elsevier Ltd All rights reserved 615 . Lys248. ization results in a lower fraction of the rhodopsin in the 303. 212. interface.. To focus on the most important structure. 307. 302. 264. we determined how retinal isomerization per. 1990). 167.. 2002). A total of 107 interactions appear results correspond to a stage in the rhodopsin cycle in Figure 5B. Saam et al. see Supplemental Data 6. 268.. were found between residue 296 (retinal) and residues ces in the transmission of the signal from retinal to the cy. 124. 93. 2001). in which each transmembrane helix was for residues 43. the b-ionone ring did not move significantly due to and hydrogen bonds are more specific in direction. we turbed the interactions described above. 189. similar results were found in previous cal. 135. 116. More strikingly. and the cytoplasmic end of helix VI (247–252). Kostenis et al. salt bridges retinal. 118. indicat. 117.204 was used. 86. As for Compared with hydrophobic interactions. 186. 178. 268. culations (Lemaitre et al. for de. 1983). D(E)RY motif from retinal is more indirect. In- from 11-cis to all-trans retinal. especially the D(E)RY motif. Glu122. This appears to be one source of the signal from retinal idues 139. 293. for details. the weakened or in- not have any obvious deviations (rmsd < 3. The first group. 178. Onrust et al. 113. 251 (Franke et al.. Rotation of the C19 methyl group was ob- lix VII (301–309).4 Å) from the cis tensified interactions tend to be clustered in certain direc- retinal crystal structure even after a 9 ns simulation with tions. and (Schenkl et al. 291. 297. 2003). instead of being randomly distributed. The interaction-energy changes between ‘‘Lys296’’ and lices III and VI between this region and retinal do not show its neighboring residues. Restraints on helix VI (254–259) (Figure 3G) or helix Procedures). restrained in the region between retinal and cytoplasmic 187. includes the cytoplasmic end of erization was distributed to several C-C single bonds of helix III (134. 211. 93. and lumirhodopsin. 293. such interdigitated to retinal are significantly altered. residue interactions coupled to retinal isomerization. relative to those from the inactive an obvious signal (Figure 3A). and the N-terminal portion of helix VIII served due to the rotation about the C8-C9 single bond. a correlation transduction is achieved by the cooperative motions of threshold of 0. the restrained dynamics trajectories. Eight indepen. (311–314). residues 83. see below. which is located its tight packing. Okada. the charge residues between bathorhodopsin. and 294–296 [retinal]) are re- strength to residue 296 (retinal) was constructed (Figures garded as significant (the underlined ones become 3B–3H) analogously to the unrestrained analysis (Fig. Figure 5A shows how the interaction (Supplemental Data 8) showed that the long-range signal network is altered by retinal isomerization. while stronger interactions resulted performed. 91.. 180. and the other ones stronger) (see Experimental ure 3A). The pathway to the meta II state. Arg135. This result is in accord with the 1997) are potential binding sites for transducin. 265. 611–623. the equilibrium fluctuations of rhodopsin in its inactive 2003. 2001. Additional simulations imental Procedures. shown in Figure 4B and Table S1. 90. 1999. which appears in about 200 fs Asp83. Seven independent MD simulations were 265.Structure Signaling Pathway of Rhodopsin ‘‘Lys296’’ (see Figure 3A). 114. the C-terminal half of he. comes more extended along its longitudinal axis due to The second group. Weaker interactions portant to investigate directly the possible role of the heli. which have smaller or no effects. including both the extracellular and cytoplasmic Local Perturbation Introduced sides of rhodopsin. as expected. and 310–313 (Bae to the protein that arises from significant alterations of et al.. in accord with the results side chain-mediated coupling of helices has been ob.. Itoh et al. Experimental studies have suggested that res. Arg147. Specifically.

May 2007 ª2007 Elsevier Ltd All rights reserved . 611–623. Structure Signaling Pathway of Rhodopsin 616 Structure 15.

117. Restraints are as follows: (B) restrained helix I (residues 56–60). (C) helix II (residues 70–82). and restrained Ca atoms are shown as red spheres. 93.5 kcal/mol plus one-fourth of its standard deviation in the ground-state simulation). because it is not visible in the chosen orientation of rhodopsin. (I) Ca atoms of helix VI (residues 254–259). Figure 3. and 294) if its interaction energy with residue 296 (including retinal) has a large difference after retinal isomerization (the deviation is larger than 0. Local Effect of Retinal Isomerization For more details. A residue is rendered as a space-filling model (86. see text). and residues having stronger interaction upon retinal isomerization (43. 264. 265. (E) helix IV (residues 158– 162). Residue 296 (including retinal) is rendered as a space-filling model in orange. respectively. 113. 291. Residue 296 (including retinal) is colored gray and is rendered as a space-filling model. response of charged residue interactions to retinal accord with Figure 5.) could make them effective as ‘‘molecular switches’’ that structure of bathorhodopsin (Nakamichi and Okada. 114. Correlation Maps of Each Residue with Residue 296. 611–623. 90. 178. 93. All figures use the same color scale with no correlation (blue) to the strongest coupling (red) (same as Figure 2A). 167. 114. see text. 207. 293. 268. 124. (A) Results for the unrestrained system averaged over 22 ns. 91. 212. (D) helix III (residues 124–132). 186. This view is from the extracellular side to the cytoplasmic side. 208. 268. (A) Diagram of retinal isomerization from 11-cis to all-trans. all residues shown have an average interaction energy with residue 296 (including retinal) larger than 1 kcal/mol in magnitude.Structure Signaling Pathway of Rhodopsin Figure 4. Including the Bound Retinal. 212. 211. (Residue 86. respond to the distant (allosteric) retinal perturbation. in 2006b). The numerical values of the interactions with significant changes are listed in Table S1. The magnitude of coupling is determined by line/row 296 of the residue correlation matrix. 116. Projected on the Inactive Rhodopsin Structure in a Ca Ribbon Representation for the Helices and Threads for the Loops (A–L) Helices. 94. and (J) Ca atoms of helix VII (residues 303–308). 289. 292. it is shown as a bond. 189. 265. May 2007 ª2007 Elsevier Ltd All rights reserved 617 . Asp83 is shown in red bonds. and 298) are colored white. certain residues. (G) helix VI (residues 254–259). otherwise. 293. 95. is not shown. 208. 118. Structure 15. 187. (B–E) Results with various restraints applied to the system during the dynamics simulation (also. 178. and the extent of the membrane are labeled. in a very new crystal isomerization was also observed. The residues having weaker interaction after retinal isom- erization (86. which has significant changes. 292. 188. 180. (L) Correlation map based on simulation with protonated Glu134. 91. (F) helix V (residues 214–226). (B) The pattern of interaction-energy deviation in the neighborhood of residue 296 after retinal isomerization. Interestingly. The Ca atoms of the conservative NPxxY and D(E)RY motifs are shown by purple and pink spheres. and 294) are colored black. (K) The correlation map based on simulation with the Asp83 side chain charge scaled down to one-fourth of its original value. 113. (H) helix VII (residues 303–308). the restrained residues are rendered in purple. 122.

Of the charged residues cited above. in response to retinal isomerization. residue 296 (including retinal) is shown as orange bonds. in the meta I 4 meta II equilibrium toward meta II 618 Structure 15. which mostly have strong correlation. For clarity. Structure Signaling Pathway of Rhodopsin Figure 5. Positively charged residues (Arg. Lys) are in red. D83N introduce a shift pected to play an important role in signal transduction. (A) Interactions with a cumulative correlation to retinal isomerization larger than 0. shielding effect than for the corresponding interactions Since they are in the interior of the portion of the in aqueous solution. VI. and the third intracellular loop (between helices V and VI) is shown in cyan. the surrounding solvent helix II and Glu122 in helix III are inside the membrane (membrane and more distant water) has a smaller between retinal and the cytoplasmic interface (Figure 3A). they are ex. respectively.. and neutral res- idues are shown by gray spheres. The interaction deviation of the D(E)RY motif clearly shows the effect of change of electrostatic energy involving Glu134.204 and an average interaction energy with an absolute value larger than 1. negatively charged residues (Asp. May 2007 ª2007 Elsevier Ltd All rights reserved . (B) An enlarged stereo view in which interactions with an average interaction energy less than 5. The Impact of Retinal Isomerization on the Residue-Residue Interactions (A and B) The rhodopsin backbone is rendered as a gray thread. are shown. that after retinal isomerization. Helices III. Strong signals are observed between charged residues at the rho- dopsin cytoplasmic interface. and its interaction with Glu247 becomes stronger (see text). Glu) are in blue. green. and blue. for a protein in a membrane. no interactions involving residue 296.0 kcal/mol in magnitude are omitted. Important charged residues identified by both this study and previous works are labeled. its interaction with Arg147 becomes weaker. The red and green dashed lines indicate interactions that become stronger or weaker. 611–623. The second intracellular loop (between helices III and IV) is shown in purple. and Ca atoms of involved residues are shown as spheres.0 kcal/mol are shown as dashed lines.e. especially at the D(E)RY motif. only Asp83 in i. respectively. Mutagenesis studies have shown protein buried in the membrane. and VII are pink.

is can be termed ‘‘tertiary allosteric coupling. spreading out isotropically and decaying with the dis- and its protonation has been shown to be necessary for tance. 1996). the coupling of conformational changes between helix VI and the C-terminal loop. this process is thought to generally involve with retinal than that calculated from wild-type simulation an allosteric transition. 298. is to related correlation factors were built with the same propagate a signal from an upstream perturbation toward protocol used in Figure 3A. 297. such the side chain charge of D83 scaled by 0. tures that were designed for signal transmission by their mation at the cytoplasmic surface without photoactivation evolutionary development. The signal-transduction pathway of GPCRs has been tions. even be amplified at the target region. This ‘‘threshold controller’’ to help in reducing the (thermal) sig. Suel et al. and 303 and weaker it has a weaker interaction correlation with retinal than in interactions with residues 297. tebrate photocycle. This suggests Based on the rhodopsin structure (Palczewski et al. following from or the NPxxY motif (Figure 3H) would significantly affect the findings described above. 87.. a 4 ns MD simulation was performed with The primary function of signal-transduction proteins. 86. Given the sensitivity of the ver- (Arnis and Hofmann.’’ Instead of highly conserved in the GPCR family (Fritze et al.e. Glu134 protonation could. The mutation D83N (DeCaluwe et al. signifi- of a displacement toward the cytoplasmic side. 1995. experiments (Farahbakhsh et al. in the the downstream partner in the signal-transduction path- simulation with the charge-scaled D83. Since the D(E)RY motif is surrounded by a large (Lockless and Ranganathan. and more generally the class of GPCRs.. When the perturbation and target sites are widely cytoplasmic side of rhodopsin have a stronger correlation separated. To further investigate the potential role that Glu134 able interactions with their neighboring residues (Fig. Asp83 has is ‘‘turned on’’ by retinal cis/trans isomerization and very weak interaction with the lipid molecules (Figure S7A). have a strong effect on the local interaction network terpreted as describing a physically connected network of and cause significant conformational changes. were found to be important in the statistical approach. 1999. might play in long-range coupling. light via retinal isomerization and. 298. Together with reduces the polarity of the side chain and weakens the re. 302. In particular.. 2003). Structure 15. 611–623.. (absolute value larger than 5 kcal/mol) in the 22 ns simula. which interacts with Glu134 (see above). in related conservation behavior of different residues was in- turn. residue 55. As in the present analysis. 299.. To evaluate this hypothesis. intrinsically coupled in their dynamics. the interac. In Fig. A possible function. of rhodopsin with protonated Glu134 (see Experimental and 303 for Asp83 (Figure 5B).. which leads to rhodopsin lar loop between helices III and IV. Spin-label coupling between retinal and the cytoplasmic interface. 2001) and the simulations. pling map with Glu134 protonated (Figure 3L) shows that actions with residues 86. Teller et al. in accord with their man eye can detect a single photon (Baylor et al.. and 299. The number of charged residues. system for investigating tertiary allosteric coupling at an As both of these interactions have relatively high energies atomic level of detail. 1995) retinal and the NPxxY motif (Figure 3L). Kuwata et al. rhodopsin sequence of the inhomogeneous nature of protein struc- with an E134Q mutation assumes a partially active confor. Retinal. the D(E)RY and NPxxY motifs tion-induced mobility change in residues on the intracellu. May 2007 ª2007 Elsevier Ltd All rights reserved 619 . as rhodopsin. The retinal residue-based cou- tion-pattern changes for Asp83 result in stronger inter. indicative the unprotonated state (Figure 3A). rhodopsin is an ideal and that between Glu134 and Glu247 becomes stronger. 1995). including residue 147.Structure Signaling Pathway of Rhodopsin (DeCaluwe et al. the D(E)RY motif moves such that the inhibited from transmitting a signal via thermal motion in interaction between Glu134 and Arg147 becomes weaker the dark-adapted state. residue-residue interactions around the D(E)RY cule optimized for signal transduction upon activation by motif showed highly ordered responses to retinal isomer. Interestingly. Thus. the signal triggers specific responses and may rhodopsin to reach the meta II state (Arnis and Hofmann. one expects that rhodopsin is a mole- ure 5B. 2003). Procedures). 1995) reported activa. The overall structural fluctuations (maximum tions (Figure 5B) suggest that anisotropic forces act on rmsd < 2. amplifies the signal. the possibility that Glu134 is a ‘‘molecular switch. which would be a factor leading to proton uptake conservation in multiple sequence alignments (MSA) by Glu134. 2000. activation. the interaction correlation analysis on rhodopsin with re- straints on it. The changes in its interac. suggested role in signal transduction. it is known experimentally that the hu. at the same time.. lowering the barrier of the inactive state / straints on the NPxxY motif (Figure 3H)..25. For monomeric proteins. As shown in Figure 3K. is that Asp83 acts as the coupling between the other motif and retinal. 1979). 302. this result sug- meta II transition and perhaps favoring the meta II state gests that perturbing either the D(E)RY motif (Figure 3L) thermodynamically. In response to retinal isomerization. and their ‘‘desolvation’’ energy is compensated by favor. like rho- (Figure 3A). we did a 4 ns simulation ure S7B). is ization. this change is expected to increase the pKa of studied by an insightful statistical analysis of amino acid Glu134.’’ which 2000. 2001). residues on the way.4 Å) are very similar to those with Glu134 unpro- Asp83. 87. Scheer et al. relative cant decreases of coupling were also observed between to helix VII. especially those on the cytoplasmic end of dopsin.... Fahmy et al. tonated (see Figure S2). suggests that the NPxxY motif and the D(E)RY motif are nal to a low level. i. Constrained by the Asp83 interactions. Glu134 in the D(E)RY motif. This feature is a con- 1995. thermal fluctuation would be less likely to overcome the energetic barriers DISCUSSION and produce a signal on the cytoplasmic side. remote regions involves tertiary structural changes and Another charged residue.

idues and the equal-time correlation function between the pairs. dues. which makes it too slow to be captured investigate further the global structural relaxation due to retinal isom- by current state of the art MD simulations. 2004). 2003) is less well defined than in signal transmission. VI and VII. For details of the system setup. the two charged residues. the coupling network inferred from the statisti. The signal propagation is expected to be a ‘‘serial. Teller et al. 2004). For tion with FRET measurements. Saam et al. to our knowledge. Nose and Klein. and Glu134..422 water molecules. of rhodopsin would be of great interest. which perturb their local electro- 2004. some of them appeared while this paper isomerization. see Supplemental Experimental Procedures 2. in accord with the experimental timescale (Rohrig et al. with each other. 2005. 2002). Although satisfactory conformational sampling of helical membrane proteins retinal isomerization is a fast process (200 fs) (Schenkl can be achieved from MD simulations (Faraldo-Gomez et al.. Therefore. we introduced a novel. A series of MD simulations was used to generate an ensemble a model of the meta II state was proposed based on an of rhodopsin structures for the analysis. coupling of interactions observed in nanosecond simula- tions to determine the actual signaling pathway that Interaction Energy Correlation The analysis is based on the energetic coupling between pairs of res- produces the allosteric transition. isomerization (Lemaitre et al. shid et al.. ment of single-molecule spectroscopy and its combina- getics and its role in generating the signaling pathway. We begin by determining the residue-residue 620 Structure 15. see Supplemental Experimental dopsin cycle. Also. with the refine- that based on the MSA. the pathway and the mechanism of 135/249. The three studies characterized the local perturbations of the retinal-binding site brought about by Molecular Dynamics Simulation retinal isomerization and the structural changes of the For the analysis of the interaction correlation (see below). in some sense. 1998) is supported by a re- cent work (Grossfield et al. see Supplemental Experimental Procedures 3. rhodopsin dark state to meta I [Ru- simulation was performed with only two residue pairs.. To study whether or not the long-range correlations observed in the the allosteric transition does not involve a large conforma- simulations are brought about by the protein scaffold. a large num- ber of coordinate sets are necessary to obtain statistically significant transmembrane helices within 10 ns or 150 ns. a ‘‘control’’ tional change (e.. 2004).. PDB ID 1HZX. crosslinking experi- that from the MD results.. 2004]). and charge-scaled Asp83 followed the procedure of the be determined by looking at its equilibrium behavior (Kar..662 atoms. starting from eight independent cis-retinal rhodopsin simulations. one simulation was continued for 9 ns with all-trans retinal. 2002). With standard analyses of simulations tion (Palczewski et al.g. Retinal isomerization was induced by imposing conformational changes) are much stronger than what is a harmonic potential with a force constant of 50 kcal/mol/radian2. Huber et al.. both the signal initiation (e. 1984. The initial coordinates of rhodopsin from microseconds to milliseconds (Menon et al. the consequential con. In actual allosteric The potential function for retinal was obtained from a published ab ini- transitions. For details. the all-trans configuration. In most cases. Zgrablic et al. restraining side chains of residues on helices a large part of the molecule... as used in these papers. tions. the system contained 138 changes of short duration were obtained. it must overcome ener. are suggested to play a special role. it was not the crystal was used in this study because it has fewer missing resi- possible to find the essential coupling involved in the rho. We showed that helices VI and VII. 2000). and their coupling via the side chains. and 24 ions. but it deals directly with the ener.g. fluorescence labels could further discussion.. which switches the C11 = C12 double bond from cis to trans in expected to be observed in equilibrium thermal fluctua- 200 fs.. This process was repeated eight times. 2001). formed. chains. Also. 94/296 and precht et al.... Moreover. 2007. or on the structural More generally. would provide additional information about the lation analysis is computationally more expensive than signal-transduction pathway. the use of multiple simulations to achieve better convergence rather formational change of rhodopsin occurs on a timescale than a single long simulation (Caves et al.. 611–623. be attached to residues on helices VI and VII to monitor A number of MD simulations investigating rhodopsin are how the rhodopsin conformation evolves after retinal in the literature. The interaction-energy corre. on the process of retinal static environment. 1997. 1983) were per- estimating the correlations between residue-residue inter.g. dues extending from the retinal-binding pocket to the which provides information about how the interactions are coupled cytoplasmic surface.6 Å resolu- Okada et al. actions in equilibrium MD simulations. Only the last three are. Asp83 was under review. 2001. 2005). To getic barriers. long-distance coupling in the actual signal propagation The NPT simulation of rhodopsin with regional restraints. The In this study. could regulate rhodopsin activity. After that. 2002. rhodopsin simulation described above. For details. as in rhodopsin. They focused on the inactive state (Cro. although some data concerning structural Procedures 1.. we identified a network of coupled resi. for details. et al.g. Hoover. thus. e. see Supplemental plus and Gao. related to the results of the EXPERIMENTAL PROCEDURES present work. 2000. ligand binding. However. a 1 ns simulation was performed with the retinal in multistep’’ process that is transmitted through the protein.. system has 34.. 5. see Supplemental Data 10. May 2007 ª2007 Elsevier Ltd All rights reserved .. at 2. were taken from the Protein Data Bank. 2003. we are able to use the Experimental Procedures 4. Grossfield et al. an experimental alanine scanning analysis changes a short time after isomerization (Crozier et al. In addition to the protein.. Rohrig et al. 2001). if erization. Recent studies indicated that elastic network normal mode (Isin et al. on the convergence these two residues function through their charged side of membrane protein simulations (Faraldo-Gomez et al. 1980. are directly involved cal analysis (Suel et al.. Since zier et al. NPT (constant pressure and constant temperature) MD simulations approach for investigating tertiary allosteric coupling by (Andersen.. latter corresponds to the equilibrium correlation between the pairs. 2005). any mutations. 2007).. 2006). i. protonated is likely to be encoded in the protein structure and can Glu134. Therefore. Structure Signaling Pathway of Rhodopsin However. results. tio quantum mechanical/molecular mechanical calculation (Tajkhor- retinal isomerization) and the remote-site response (e. Chain A of the dimer in on the MD timescale. To overcome DOPC molecules.e. the entire this limitation. 2007)... the former appears to involve ments.

Responses of retinal relation matrix is obtained by summing over all interacting pairs involv- rods to single photons. the nonbonded interaction energy. K.E. Mapping of contact sites in complex formation between transducin and light-activated rhodopsin X N N  X   I. RCI. J. 17 (Roma: Academia Nationale dei Lincei). (2) 611/DC1/. and Woolf. (2001).t l  Ek. Chem.C.structure. Andersen. the membrane lipids. ACKNOWLEDGMENTS nate set t. The dimension of the residue scale conformational change.S. which are likely to be important Brunori. l Accepted: April 6. P. 559–574.S. Caves.t l  Ek. the residue correlation between 10 and 17 would be 0.j and k. Protein Sci. 2007 Pf t=1 Ei.P. retinal was treated values to residue 296 independently calculated from two sets of coor- as part of the side chain of residue 296.elec vdw j + Ei.. Stevens. l . by molecular dynamics calculations. Ci. K. (2005).K. H.A.E. H. 2384–2393. (1998). which generates a Bae.mjn J is 0. G. J. duced in addition to the interaction-energy cutoff (see Figure S3). J. the correlation between residues I and J.35) is Crozier. Pf  t   t = 1 Ei. if the correlation between interaction pairs (10. Graber. Biol. RCI.. the PME potential was not in- perturbation of distal residue interaction by retinal isomerization was cluded here because it is expected to provide only a small correlation calculated. Evanseck..G. since it includes indirect interactions. of course.. For example. is defined as Effects of Retinal Isomerization Ei. between coordinates into two sets. T.. this means that the resi. and Karplus. Stevens. which were generated by randomly partitioning the recorded frame (see below). is defined as Cai. Molecular determinants of selectivity in 5-hydroxy- matrix to a reasonable value. (4) m=1 n=1 Acad. Only amino acids The error estimates of the values in Figure 3A are shown in Figure S8. i.M. Flood. S. 0. J. and Schachman. L.D. that involving change in retinal leads to G-protein activation: initial events suggested retinal) of the residue correlation matrix on the rhodopsin structure.. J  by covalent crosslinking: use of a photoactivatable reagent. Natl. j  Ei. Matrix Assembly Arnis.t j  Ei. N. respectively. Anderson.S. In 32071–32077. and Woolf. M.D. for distant residues... Molecular dynamics simulations at constant pressure and/or temperature. otherwise. (1999). l Received: December 13. Science 308. S.2 ps.J. 333.J.. i. S.e.org/cgi/content/full/15/5/ E i.. K..23 kcal/mol. Forrest. Sci. J = Cmjn 3 dmjn . 272. T. and Hamm. (2005). 0) to 79. Biol. pairs that are neighbors in sequence are not included because they are covalently bonded. Depree.l = r ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2  2ffi : (3) Revised: March 30. P. It is useful to H. Proteins 66. Chem. a correlation cutoff (Ccutoff) was intro. (1) The isomerization of retinal.l. Allosteric mechanisms of correlation between interactions m and n obtained from the interaction signal transduction. 993 3 993 interaction correlation matrix (Figure S4).P... Allosteric Proteins: 40 Years with Monod-Wyman-Changeux. tryptamine1B receptor-G protein interactions. j ðji  jj>1Þ.. which is essentially a projec. (1997). and Yau. 7. The potential the main chain and side chain atoms. 1424–1428. plicit membrane bilayer: coupling between local retinal and larger due correlation can be greater than unity. pling with 5-hydroxytryptamine1B receptors. L. correlation matrix is equal to the number of residues in rhodopsin (348). 14963–14971. 274. tions of crambin. However. the absolute value of the average interac- tion energies varies from 5. Locally accessi- ble conformations of proteins: multiple molecular dynamics simula- where dI.25j17.e.. tion of the interaction energy correlation matrix on the residue space.W. umns and rows corresponding to all nonsequential neighboring resi. (1979). projecting the changes of retinal-involved interactions on to the pairwise interaction energy between residues within a single the interaction correlation matrix. Photoregeneration of bovine rho- From Equation 3. and Hofmann.35 and that between pairs (10. Skiba. I and J. M. dI. details are given in Supplemental rhodopsin molecule. and f is the number of coordinate sets. j = Ei. L. 493–514. is defined as Institutes of Health.A. tions m and n or n and m. J. In the pres.e. (2003). correlation matrix. This is. Bae. Y. D.70 due Molecular dynamics simulation of dark-adapted rhodopsin in an ex- to these two interaction-energy correlations. different from the direct I. 611–623.G. as part of residue 296.P. f t = 1 i. To reduce the size of the Graber.. water mole. Phys. M. Careri. the Ccutoff was set to 0. 649–666. were included in the analysis. REFERENCES ent simulation of rhodopsin. T.elec j and Ei. 613–634.j.J.vdw j are the electrostatic and van der Waals interac- However. K. H. and ions were not considered. If the dimension of the interaction correlation ma. i and j. H. Biochemistry 34.B.. H. we mean that for any pair of residues. Itoh.85 3 108 (i. J.. and Khorana. M. the effect on the interactions between more distal residues is tion energies. between residues i and j. Cmjn is the Changeux. as stated above. S. j Ek.J.30) is 0. jjk..J interaction. where Ei. The average interaction energy between resi- Supplemental Data due i and residue j is defined as Supplemental Data include one table and Experimental Procedures 1X f and are available at http://www. 2006 Ci. 9333–9340. K..P.B. Lamb. j Ek. Crozier. (1995). j where Ei. the interaction-energy correlation matrix has col. 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