Photoactivation Intermediates of a G-Protein Coupled Receptor

Article pubs.acs.org/JPCB

Photoactivation Intermediates of a G‑Protein Coupled Receptor Rhodopsin Investigated by a Hybrid Molecular Simulation Motoshi Kamiya† and Shigehiko Hayashi* Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan S Supporting Information *

ABSTRACT: Rhodopsin is a G-protein coupled receptor functioning as a photoreceptor for vision through photoactivation of a covalently bound ligand of a retinal protonated Schiff base chromophore. Despite the availability of structural information on the inactivated and activated forms of the receptor, the transition processes initiated by the photoabsorption have not been well understood. Here we theoretically examined the photoactivation processes by means of molecular dynamics (MD) simulations and ab initio quantum mechanical/molecular mechanical (QM/MM) free energy geometry optimizations which enabled accurate geometry determination of the ligand molecule in ample statistical conformational samples of the protein. Structures of the intermediate states of the activation process, blue-shifted intermediate and Lumi, as well as the dark state first generated by MD simulations and then refined by the QM/MM free energy geometry optimizations were characterized by large displacement of the β-ionone ring of retinal along with change in the hydrogen bond of the protonated Schiff base. The ab initio calculations of vibrational and electronic spectroscopic properties of those states well reproduced the experimental observations and successfully identified the molecular origins underlying the spectroscopic features. The structural evolution in the formation of the intermediates provides a molecular insight into the efficient activation processes of the receptor.

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INTRODUCTION Rhodopsin (Rh) in the vertebrate retina is one of the primary photoreceptors for vision.1,2 The visual rhodopsin is classified as a member of G-protein coupled receptors (GPCRs) which constitute a large family of membrane proteins responsible for receptions of external signals and initiations of intracellular signaling cascades in cellular signal transductions, and thus, many of them have been targeted for drug discovery.3 Rhodopsin consists of seven transmembrane helices, called opsin, as seen in other GPCRs and covalently binds a retinal at a lysine residue through a protonated Schiff base linkage which serves as a chromophore to detect light (Figure 1a). In the resting state, Rho, in the dark, the retinal protonated Schiff base resides in opsin in 11-cis conformation and acts as a strong inverse antagonist which significantly suppresses a signaling noise due to thermal activation4 (Figure 2). The photoabsorption of the retinal chromophore turns its 11-cis conformation into an all-trans one through photoisomerization with a high quantum yield of 0.67,5 and eventually leads to a Gprotein activating state, Meta-II, with an agonist of the all-trans deprotonated form of the chromophore through efficient conformational relaxation on the millisecond time scale6 (Figure 2). The strong inactivation in the dark, the high quantum yield of the primary photochemical reaction, and the following highly efficient conformational relaxation to the active form make the receptor an almost single photon counter. © XXXX American Chemical Society

Since an X-ray crystallographic structure of Rh in the dark was solved as the first atomic model of GPCR,7 many X-ray crystallographic structures of various GPCRs including agonist-, antagonist-, and inverse agonist-bound forms as well as apo ones have been determined.8 Those structures have elucidated in atomic detail molecular mechanisms of the activation of GPCRs such as cytoplasmic opening of Helix VI hinged at a well-conserved tryptophan accompanied by breakage of the socalled “ionic lock” which is a salt bridge between well-conserved residues located in Helixes III and VI. However, the atomistic view of the coupling between the ligand molecules and the protein conformational changes activating G-proteins is still obscure due to the lack of a clear understanding of the transition dynamics connecting between the inactive state and the active one. Rh is an ideal GPCR to examine the transition dynamics, because the ligand is not diffusive but is covalently bound in the protein during the transition from the inactive state to the active one, and the transition can be initiated by light illumination, enabling precise spectroscopic and structural measurements of the conformational transitions. In fact, several intermediate states, Batho, Special Issue: Klaus Schulten Memorial Issue Received: December 30, 2016 Revised: February 12, 2017

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DOI: 10.1021/acs.jpcb.6b13050 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. Structures of Rh. (a) Structure of the Rho dark state. Flexible regions in the N and C terminal regions are omitted for clarity. The protein is drawn in the orientation where the cytoplasmic side and the extracellular one are located at the top and the bottom, respectively. An 11-cis retinal protonated Schiff base chromophore bound to Lys296 is depicted in licorice representation. Helixes III and IV are drawn transparently. (b) X-ray crystallographic structure of the Rho dark state54 (PDB ID: 3C9L) and the Lumi intermediate one15 (PDB ID: 2HPY). A view from the cytoplasmic side. Helixes IV and VI of the Rho and Lumi states are drawn in green and pink, respectively. The chromophores in the Rho and Lumi states are depicted in the standard color code and in pink, respectively. Trp265 and Ala168 in the Rho state are drawn. (c) A QM/MM simulation system for the Rho state in a periodic boundary condition. Quantum mechanically treated molecules are depicted in van der Waals representation.

temperature does not move significantly from the position in Rho and stays far away from Ala169 in Helix IV15 (Figure 1b). The discrepancy between the experiments of the photoactivated species might arise from the difference in sample conditions. Especially, the crystalline condition tends to suppress conformational changes by the photoactivation. For example, light illumination of Rh crystal that produced the deprotonated form of the chromophore as seen in Meta-II16 led to conformational changes much smaller than those observed in X-ray crystallographic structures of the active form of opsin and Meta-II obtained by soaking of the opsin crystals with all-trans retinal.17 It is therefore important to examine the photoactivated conformational dynamics in a membrane environment. Molecular dynamics (MD) simulations of membrane proteins in an explicit membrane environment allow one to investigate in atomic detail the conformational dynamics of the photoactivated proteins. Schulten is one of the pioneers of MD simulations of membrane proteins and has been making significant contributions to great advances in the research field.18,19 A key of the successful molecular simulations of the photoactivated membrane proteins is accuracy in the description of the nonequilibrium processes of the complex molecular systems. For example, a seminal simulation study of a hybrid quantum mechanical/molecular mechanical (QM/MM) method on the photoactivation of Rh by Warshel with crude approximations of empirical treatment of electronic wave functions of the chromophore and of removal of atomic details of the surrounding environment predicted the “bicycle pedal” corotations of C11C12 and C15N16,20 which is unfortunately not supported by most of the experimental and computational evidence obtained so far. Then, later improvement of the empirical retinal models by the researcher and coworkers21,22 successfully provided a structural model of the photoproduct which does not involve the bicycle pedal corotation of C11C12 and C15N16 but does partial twisting of other bonds, qualitatively agreeing with those obtained by more sophisticated calculations carried out recently (see below). Furthermore, the fully atomistic treatment of the membrane protein, thanks to the availability of the X-ray crystallographic

Figure 2. Scheme of photoactivation processes of rhodopsin.6,86

BSI, Lumi, and Meta-I, during the photoactivated transition to the G-protein activating state, Meta-II, have been well characterized spectroscopically (Figure 2). Resonance Raman,9 Fourier transformed infrared (FTIR), 10,11 and NMR12,13 studies uncovered various molecular events in the photoactivation processes in terms of the conformations of the chromophore and hydrogen-bond strength of the Schiff base. Nevertheless, a consensus of the conformational changes of the photoactivated ligand and its structural coupling with the protein has not been obtained. For example, a cross-linking experiment with a 3-diazo-4-oxo-retinal analogue chromophore14 showed that the β-ionone ring of the chromophore undergoes significant conformational changes upon the formation of Lumi; it departs from a position in Rho, which is close to the well-conserved tryptophan, Trp265, in Helix VI, and approaches Ala169 in Helix IV. On the other hand, the βionone ring in an X-ray crystallographic structure of Lumi generated by light illumination of Rh crystals at low B


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chromophore in the protein leading to the G-protein activating state.

structures, and advance in hybrid simulation techniques employing ab initio quantum chemistry methods for the description of the complex electronic structure of the chromophore have enabled researchers to uncover characteristic molecular interactions and dynamics underlying experimental observations.23−42 For example, Schulten and coworkers performed the excited state ab initio QM/MM MD simulations of the primary photoisomerization reactions of retinal proteins26,36 and clearly characterized and furthermore successfully predicted experimentally observed time-resolved spectroscopic signals.38,43 They also carried out ab initio QM/ MM calculations to examine early intermediate states of a photoactivated retinal protein and successfully identified molecular conformational changes and interactions responsible for the storage of the photon energy utilized for its function.25,28 In those studies, the accurate calculations of spectroscopic properties such as vibrational and electronic photoabsorption spectra enabled by the ab initio QM/MM calculations were of crucial importance in obtaining the high reliability and predictability of the simulations. In fact, theoretical assignment of vibrational spectral changes upon formation of an early photoactivated intermediate of a retinal protein28 was later confirmed by an experimental study,44 reliably providing a novel insight into the molecular mechanism of the photon energy storage. For the photoactivation process of Rh, Schulten and coworkers45 and other researchers41,46−51 carried out classical MD simulations for the fully solvated and membrane embedded systems. Those MD simulations on the nano- to microsecond time scales showed that the β-ionone ring undergoes large positional displacement in thermal relaxation after photoisomerization, consistent with the cross-linking experiment mentioned above. Campomanes et al.41 further analyzed photoabsorption spectra of the early intermediate states of which conformations were sampled by short QM/MM MD simulations (5 ps each). However, the theoretical characterizations of the intermediate states are still obscure due to lack of accuracy of the classical force field of the chromophore possessing the complex electronic structure to describe its highly distorted conformation generated upon photoactivation in classical MD simulations and lack of sufficient statistical conformational samples to stably represent the intermediate states formed in the conformational relaxation on the sub-microsecond time scale. In the present study, we theoretically investigated the photoactivation process of Rh through modeling of the early intermediate states, BSI and Lumi, with the QM/MM RWFESCF free energy geometry optimization method.52,53 The method enables one to accurately optimize the molecular geometry of the conformationally strained chromophore and protein groups strongly interacting with it upon photoactivation at the ab initio quantum chemical level of theory on free energy surfaces of the surrounding protein environment constructed with ample statistical conformational samples obtained by classical MD simulations. The high accuracy of molecular structures and interactions and the statistically well-defined treatment of the intermediate states allowed us to simultaneously characterize both electronic and vibrational spectroscopic signals, clearly illustrating the molecular events underlying the formations of the intermediate states. The theoretical characterization of the conformational changes in the early stages of the photocycle provides a mechanistic insight into the efficient formation of the agonist form of the ligand

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METHODS Simulation System. The initial structure of bovine rhodopsin was built from the dark state (Rho) X-ray structure by Stenkamp (PDB ID: 3C9L).54 The simulation system in a periodic boundary condition included 108 POPC, 12,411 water molecules, and 2 sodium ions in addition to the protein (Figure 1c). Asp83 and Glu122 were set to be protonated,55 and Glu181 was to be deprotonated.56,57 The initial conformation around the C6−C7 bond of retinal was in a cis form, following experimental evidence of a NMR study.13 We employed the CHARMM22/CMAP force field for the protein and the ions and CHARMM27 for the lipid molecules.58−61 The TIP3P model was used for water molecules. The force field of retinal was taken from one employed in previous studies on microbial rhodopsins40 except for the dihedral angle around the C6−C7 bond. The 6-cis form of the retinal was found to be highly unstable with the original force field and to be easily turned into the 6-trans form even in the binding pocket in the dark state, contradicting the experimental evidence.13 We therefore modified the force constants of the dihedral parameters around the C6−C7 bond from 1.405 to 3.0 kcal/mol and C1−C6 from 0.0 to 0.2 kcal/mol. Although these parameters were determined somewhat ad hoc, all of the structures obtained with the modified force field were later refined at the highly accurate ab initio QM level of theory as described below. MD simulations were performed with NAMD.62 Molecular images in figures were created with VMD.63 C−H bond order parameters of the lipid molecules in the membrane (Figure S1 in the Supporting Information) indicate that the membrane did not fall into a gel-like state in the current settings. Equilibration. The simulation system was first energy minimized and then equilibrated for 2 ns in NVT condition at 100 K and further 2 ns in NPT condition at 1 bar and 100 K. The temperature was gradually raised up to 300 K in 2 ns. The system was then relaxed in NPT condition at 300 K for 2 ns, followed by equilibration for 50 ns in NVT condition. Isomerization. Isomerization of the 11-cis form of retinal to the all-trans one in the formation of the first photointermediate, Batho, was performed by classical MD calculations, where the potential periodicity of the dihedral angle around the C11−C12 bond was changed from a double-well potential having minima in both of cis and trans conformations to a single-well potential having a minimum only for the trans. We obtained several isomerization trajectories for 1 ns each. We confirmed that a salt-bridge between the Schiff base of the chromophore and Glu113 remained intact upon the formation of Batho in almost all of the trajectories, consistent with experimental9 and quantum chemical45 studies, despite the crude treatment of the isomerization simulation. One trajectory with the intact salt-bridge in the trials was followed by a trajectory calculation with the reset original potential function around the C11−C12 bond for ∼300 ns. We selected some snapshots from the trajectory which were representative of BSI and Lumi intermediates and were subject to the structural refinement by ab initio QM/MM free energy geometry optimization described below. QM/MM Free Energy Geometry Optimization. Molecular structures of BSI and Lumi intermediates as well as the Rho dark state were refined by QM/MM RWFE-SCF geometry optimization. Methodological details are described elseC


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The Journal of Physical Chemistry B Table 1. Dihedral Angles of a Polyene Chain of a Retinal Protonated Schiff Base Chromophore (deg)a C6C7 C7C8 C8C9 C9C10 C10C11 C11C12 C12C13 C13C14 C14C15 C15Nζ a

Rho

3C9L54

BSI

Lumi(1)

Lumi(2)

2HPY15

−46.4 (46.4) −177.5 (2.5) 172.2 (7.8) 176.1 (3.9) −177.8 (2.2) −7.7 (7.7) 156.4 (23.6) −174.2 (5.8) 169.0 (11.0) 175.5 (4.5)

−59.2 (59.2) −180.0 (0.0) −173.7 (6.3) 179.4 (0.6) 171.9 (8.1) −2.5 (2.5) 167.3 (12.7) 178.7 (1.3) −178.8 (1.2) 174.6 (5.4)

−40.1 (40.1) −175.0 (5.0) 177.6 (2.4) −176.5 (3.5) 175.1 (4.9) −170.4 (9.6) 171.4 (8.6) −172.2 (7.8) 173.1 (6.9) −177.2 (2.8)

−44.0 (44.0) −172.3 (7.7) 179.9 (0.1) −172.2 (7.8) 174.7 (5.3) −166.0 (14.0) 171.2 (8.8) −169.8 (10.2) 178.5 (1.5) −179.8 (0.2)

−42.7 (42.7) −174.8 (5.2) −179.9 (0.1) −172.3 (7.7) 174.9 (5.1) −171.0 (9.0) 176.9 (3.1) −177.6 (2.4) −177.8 (2.2) −176.1 (3.9)

−5.2 (5.2) −177.9 (2.1) 164.4 (15.6) −161.5 (18.5) 168.6 (11.4) −157.7 (22.3) −168.2 (11.8) −176.4 (3.6) −177.4 (2.6) −165.8 (14.2)

Values in parentheses are deviations from the planarity (0 or 180°).

where.52,53 Briefly, the molecular geometry of a quantum mechanically treated region is optimized on a mean field free energy surface constructed with conformational samples obtained by a classical MD simulation for the surrounding environment treated with MM force fields. Iteration of a cycle of the QM/MM free energy geometry optimization and the MD conformational sampling called the sequential sampling is performed until the convergence well-defined with a statistical reweighting scheme is achieved. The method is designed to be fully variational and highly efficient by the reweighting treatment and complete separation of the procedures of the QM/MM geometry optimization and the classical MD sampling with MM force fields. The QM regions are depicted in Figure S2 in the Supporting Information. The common QM region of the Rho dark state and BSI and Lumi intermediates contains retinal protonated Schiff base linked to Lys296, the side chain of Glu113, a water molecule hydrogen-bonded to Glu113, and the backbone C O group of Ser186 and NH group of Cys187. The QM region of BSI additionally includes the side chain of Ser186, which was hydrogen-bonded to Glu113. In two representative structures of the Lumi state, Lumi(1) and Lumi(2), another water molecule which was absent in Rho and BSI and appeared near Glu113 in the formation of Lumi was added to the QM region. The side chain of Thr94 was also added to the QM region of Lumi(2). The QM−MM boundaries were set to the CδCε bond for Lys296, CβCγ bond for Glu113, CαC and NCα bonds for the peptide group of Ser186 and Cys187, NCα bonds for Ser186 for BSI, and CαCβ bond for Thr94, respectively. The boundaries of the QM region were capped with hydrogen atoms. The electronic wave functions of the QM regions were obtained with density functional theory (DFT) calculations with the B3LYP functional. The basis sets employed were 631+G(d,p) for the carboxyl group of Glu113 and the water molecules hydrogen-bonded to it, 6-31G for the boundary methyl group involving link atoms, and 6-31G(d,p) for the others. The MD simulation in each cycle of the sequential sampling consists of trajectory calculations for an equilibration and a conformational sampling for 2 ns, respectively, for Rho and BSI, and those for 0.96 ns, respectively, for Lumi(1) and Lumi(2). The reweighted MM mean fields were constructed with 20,000 MM conformational samples from the trajectory of the conformational sampling for the former states, and with 32,000 samples for the latter ones, respectively. The free energy geometry optimizations for Rho, BSI, Lumi(1), and Lumi(2) required 81, 24, 40, and 69 cycles of the sequential

samplings (MD simulations for 324, 96, 76.8, and 132.48 ns in total), respectively. Calculation of Spectroscopic Properties. Vibrational and electronic spectroscopic properties of the intermediates as well as the dark one were evaluated by the QM/MM calculations. Vibrational normal modes were evaluated with Hessian matrices of the free energy surfaces obtained by QM/ MM RWFE-SCF calculations.52,53 The vibrational frequencies were scaled by the generic scaling factor for the DFT calculation at the B3LYP/6-31G(d,p) level of theory, 0.961.64 The single point excitation (photoabsorption) energy calculations were performed at the three-states-averaged XMCQDPT2/CASSCF(12,12)/6-31G(d,p) level65,66 at the QM/MM RWFE optimized geometries with the MM electrostatic mean fields. The CAS space includes all of the valence πorbitals and π-electrons of the retinal chromophore. To reduce the computational cost of the highly demanding multireference perturbation calculations, only the chromophore and Lys296 with the QM−MM boundary at the Cδ−Cε bond were taken as the QM region of the excitation energy calculation. The atoms included in the QM regions in the free energy geometry optimization but not in the QM region in the excitation energy calculation were treated as the MM atoms with the effective point charges of the MM force fields. In the QM/MM treatment, quantum contributions from the surrounding environment such as electronic polarization and charge transfers67,68 were neglected. We therefore focused on relative excitation energies with respect to the dark state where cancellations of the possible errors are expected. Valsson et al. showed that the quantum effect of the counterion carboxylate of Glu113, which could largely influence the spectral shift, is not significant and the spectral shift can be qualitatively described with QM−MM electrostatic interaction.69 Although quantum interactions with more remote surroundings would give rise to a large change in the excitation energy due to their larger size, cancellations of the relative excitation energies can be expected because of the nonspecific and short-range nature of those interactions. The electrostatic contributions of the surrounding environment to the excitation energies were analyzed by decomposition as performed in previous studies.23,24,40,70 First, the electrostatic contributions were decomposed into those of the protein and the others (water molecules, ions, and the membrane lipid molecules) in the simulation box. The protein contributions were further decomposed into contributions of the individual residues. Effective point charges of the chromophore atoms derived from the CASSCF(12,12)/6D


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The Journal of Physical Chemistry B 31G(d,p) wave function were used to calculate the electrostatic contributions.

The hydrogen-bond strength between the protonated Schiff base and Glu113 was assessed by vibrational frequency calculations with normal-mode analysis of the free energy surface. The vibrational frequency of the N−D stretching mode of the protonated Schiff base was evaluated to be 1989 cm−1 with the generic scaling factor,64 0.961 (Table 2), which is in

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RESULTS Rho Dark State. Before examining the photoactivation process, the molecular structure and spectroscopic properties of the Rho dark state obtained by the QM/MM RWFE-SCF method are briefly surveyed. The ab initio treatment of the QM molecules and the sufficient conformational sampling of the MM environment enabled electronically and statistically highly accurate refinement of the active site. Table 1 summarizes the optimized dihedral angles of the polyene chain of the chromophore. A large deviation from the planarity by 23.6° arose around the C12C13 bond, while torsion around the C11C12 bond in the cis conformation remained modest; the deviation is 7.7°. The torsional characters of the polyene chain are somewhat different from X-ray crystallographic models, where torsion around the C12C13 bond is smaller (deviations from the planarity are 8.5° for 1L9H (PDB ID),71 6.5 and 1.4° for models A and B of 1U19,30 and 12.7° for 3C9L,54 respectively), and those around the C11C12 bonds of 1U19 are larger (deviations by 40.8 and 36.1°). In a previous ab initio QM/MM geometry optimization27 with a protein model fixed at an X-ray crystallographic structure72 and a QM/MM MD simulation with a semiempirical QM method,30 torsions around the C12C13 bond and the C11C12 bond are also smaller (by ∼10°) and larger (by ∼16−18°). The torsions around the C11C12 and/or C12C13 bonds appear because the Schiff base half of the polyene chain and the β-ionone one need to mutually orient slightly differently in order to accommodate the V-shape structure of the chromophore in the 11-cis conformation in the binding pocket. The present structural refinement at the ab initio QM level of theory in the sufficiently thermally relaxed MM conformations showed that the mutual orientational difference between those halves is achieved by the torsion around the softer C12C13 single bond than the C11C12 double bond. It should, however, be noted that, although the present approach significantly improved computational accuracies in terms of both electronic structures and statistical sampling, quantitative comparisons with other theoretical and experimental results are still difficult because of limitations of the computational30,73−75 and experimental76−79 techniques. A marked difference in the interaction of the protonated Schiff base with its counterion of carboxylate of Glu113 between the X-ray crystallographic models and the present refined structure was found. In the X-ray crystallographic structures,30,54,71 the distance between Nζ of the Schiff base and the closest oxygen atom of Glu113 is >3.0 Å, which is longer than the distance of a hydrogen bond in a direct salt bridge. The ab initio QM/MM geometry optimization with a fixed protein model27 also gave a similar distance (3.3 Å). On the other hand, the distance determined at the ab initio QM level of theory with the sufficient thermal MM conformational relaxation of the protein environment in the present study provided a strong hydrogen-bond formation in a direct saltbridge with a distance of 2.67 Å, which is consistent with a previous study by QM/MM MD simulation.30 The zwitterionic state of the direct salt-bridge is stabilized by Thr94 and a water molecule in the vicinity of the salt-bridge, while the zwitterionic state is unstable without the surroundings, as suggested by Buß and co-workers.80

Table 2. Shifts of excitation energies, ΔΔES1−S0, and Vibrational Frequencies of the Nζ−D Stretching Mode of a Protonated Schiff Base, νN−D νN‑Da (cm−1)

ΔΔES1−S0 (eV) Calc. Rho BSI Lumi(1) Lumi(2) a

0.0 (2.33) 0.21 −0.01 0.01

Expt. b

87

0.0 (2.49) 0.11 0.03

b

Calc.

Expt.10

1989 1986 2328 2388

1967, 2012 n.a. 2337

Scaled by 0.961. bAbsolute values of excitation energies.

good agreement with the experimentally observed frequencies,10 1967 and 2012 cm−1. Given also that the experimental frequencies of Rho are low among other rhodopsin proteins,81,82 the protonated Schiff base is likely to form a direct strong hydrogen bond with Glu113. The photoabsorption energy of the chromophore was also computed for the free energetically optimized model with the electrostatic mean field of the MM conformational samples obtained by a MD simulation (Table 2). The excitation energy was evaluated to be 2.33 eV, which is in qualitative agreement with the experimental one, 2.49 eV, although the computed value is slightly underestimated presumably due to approximation of neglecting quantum mechanical interactions, such as polarization and charge transfer, of the protonated Schiff base with Glu113 introduced in the calculation of the excitation energy to reduce the highly demanding computational cost of the multireference perturbation calculations (see Methods). BSI State. In short time after the isomerization of the chromophore, structural changes were limited to a local region around the isomerizing bond, and other significant conformational changes were not observed in most of the trajectories. Then, in the following few nanoseconds of the classical MD simulations, the β-ionone ring of the chromophore flipped toward helix IV while the salt-bridge of the Schiff base with its counterion of Glu113 was kept intact and the 6-s-cis conformation of the β-ionone ring was maintained. The observed conformational changes are consistent with previous MD simulations.41,45,46,50,51 To refine and characterize the newly formed conformation after the significant conformational change of the β-ionone ring, which is assigned to the BSI state as described below, we carried out a QM/MM RWFE-SCF free energy geometry optimization from a snapshot of the classical MD simulation. Parts a and b of Figure 3 illustrate the conformational changes of the retinal binding pocket. The flip of the β-ionone ring was accompanied by straightening the polyene chain in the all-trans conformation. In fact, any large torsion of its dihedral angles was not observed (Table 1). The β-ionone ring was detached from Trp265 and then accommodated in a region surrounded by Thr118, Cys167, Ala168, Phe203, and His211. The space in the vicinity of Trp265 originally occupied by the β-ionone ring in Rho was filled by Phe212. In contrast to the large movement of the β-ionone ring, conformational changes in the Schiff base half of the E


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Figure 3. Conformational changes in photoactivation processes obtained by theoretical calculations. (a) Views of the retinal binding site from the cytoplasmic side. (b) Molecular structures of the chromophore in Rho (green), BSI (gray), and Lumi(2) (pink) states, respectively. (c) Comparison of the retinal binding sites of Rho (green) and Lumi(2) (pink) states, respectively.

chromophore were modest. The strong hydrogen bond of the protonated Schiff base with Glu113 was maintained; the Nζ−O distance between them is 2.67 Å, which is the same as that in the Rho state. Vibrational frequency of the N−D stretching mode of the protonated Schiff base obtained by the normalmode analysis, 1986 cm−1, was also kept similar to that of the Rho state, 1989 cm−1 (Table 2), indicating that the strength of the hydrogen bond was not altered upon the formation of the intermediate. The computed electronic photoabsorption energy, on the other hand, remarkably increased by 0.21 eV (Table 2). Because of the blue shift of the absorption maximum, the intermediate state is assigned to the BSI state (Figure 2). One of the origins of the blue shift is electrostatic interaction of the chromophore with the protein surroundings. Table 3 shows

Figure 4 shows the electrostatic contributions of the individual residues of the protein. The main contributions are

Table 3. Electrostatic Contributions of the Surrounding Environment to Excitation Energies (kcal/mol)a

Figure 4. Electrostatic contributions of individual residues of the protein to excitation energies of Rho, BSI, Lumi(1), and Lumi(2) states (see Methods). Positive (negative) values correspond to contributions of blue (red) shifts of the excitation energies.

Rho BSI Lumi(1) Lumi(2) a

total (protein + water + membrane + ions)

protein

water + membrane + ions

12.63 16.53 12.27 10.93

11.58 14.04 11.37 7.91

1.05 2.49 0.91 3.02

found to originate from Glu113 and Thr94, which locate in the vicinity of the protonated Schiff base. Although movement of Glu113 and Thr94 upon the formation of the BSI state was modest, their side chains underwent slight conformational changes (Figures 3a and 5a). Rotation of the carboxylate group of Glu113 accompanied by formation of a hydrogen bond with the hydroxyl group of Ser186 was observed. The rotation slightly displaced the negatively charged oxygen atom, which is the one not hydrogen-bonded with the protonated Schiff base, toward the Schiff base side, increasing its electrostatic coupling with the positive charge migration from the protonated Schiff base toward the β-ionone ring side along the polyene chain upon photoexcitation,23,24,40,70 thus producing the contribution of the blue shift. The rotation also induced switch of a hydrogen-bond partner of the hydroxyl group of Thr94 from the carboxylate of Glu113 in the Rho state to the main chain carbonyl group of Cys185 which diminished the electrostatic coupling of the hydroxyl group of Thr94, giving the contribution of the red shift in the Rho state. Contribution of structural changes of the chromophore to the excitation energy was also examined at the optimized geometry without the electrostatic field of the surrounding

Further details can be found in the Supporting Information.

analysis of the electrostatic contribution to the excitation energy (see Methods). The contribution of the nonprotein moiety was small in all of the states. More detailed analyses are summarized in Tables S1 and S2 in the Supporting Information. The small contributions of the internal water molecules are attributed to the fact that no water molecules were directly attached to the retinal Schiff base. Upon the formation of the BSI state, the electrostatic contribution largely increased by 3.9 kcal/mol, although the contribution is expected to be slightly overestimated due to the use of the CASSCF wave function for the analysis (see Methods). The increase of the electrostatic contribution is decomposed into those of the protein by 2.4 kcal/mol and of the water molecules, ions, and the membrane lipid molecules in the simulation system by 1.4 kcal/mol. F


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states, which are assigned to be Lumi intermediates, Lumi(1) and Lumi(2), as described below. The highly accurate structural refinement of Lumi states allowed us to identify subtle but significant conformational changes from the BSI state. First, the β-ionone ring was further displaced toward Helix IV and approached Cys167. The close proximity of the β-ionone ring to Cys167 in Lumi states is consistent with the experimental evidence of their cross-linking in Lumi states.7 Second, the Nζ−O distance of the hydrogen bond between the protonated Schiff base and Glu113 was significantly elongated from 2.67 Å in Rho and BSI states to 2.95 Å in Lumi(1) and 2.99 Å in Lumi(2). The hydrogen bond is therefore weakened upon the formation of Lumi states. The elongation of the hydrogen bond originates from the slight rotation of the chromophore accompanied by the displacement of the β-ionone ring described above; the rotation made the Nζ−H bond of the protonated Schiff base point toward the extracellular side, and consequently induced mismatch of the mutual orientations of the protonated Schiff base and the carboxylate of Glu113 for the hydrogen-bond formation (Figure 5b). Vibrational frequency of the N−D stretching mode of the protonated Schiff base was computed to be upshifted to 2328 cm−1 in Lumi(1) and 2388 cm−1 in Lumi(2), which are in good agreement with FTIR experimental evidence10 (Table 2). The weakening of the hydrogen bond described above well explains the upshift of the vibrational frequency. Photoabsorption energies of Lumi states were also calculated and found to be red-shifted from the BSI state and to be similar to the Rho state. The red shift from BSI is mainly attributed to a decrease of the blue-shifting electrostatic contributions from the surrounding environment by 4.2 kcal/mol for Lumi(1) and 5.6 kcal/mol for Lumi(2) (Table 3), especially from Glu113 (Figure 4), consistent with the weakening of the hydrogen bond between the protonated Schiff base and Glu113. The structural changes of the chromophore also contributed to the red shift to some extent (Table 4).

Figure 5. Close up views of the protonated Schiff base region. (a) Comparison of structures in Rho (green) and BSI (gray) states, respectively. (b) Comparison of structures in BSI (gray) and Lumi(1) (purple) states, respectively.

environment with the same computational scheme as that used for the protein (Table 4). The excitation energy of the Table 4. Excitation Energies of a Retinal Protonated Schiff Base Chromophore without the Surrounding Environment, ΔES1−S0,gasa ΔES1−S0,gas (eV) Rho BSI Lumi(1) Lumi(2)

2.04 2.14 2.08 2.07

(0.00)b (0.10) (0.04) (0.03)

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DISCUSSION In the present study, BSI and Lumi intermediates as well as Rho dark state were theoretically modeled by the classical MD simulations followed by refinement with the QM/MM free energy geometry optimizations. After the isomerization of the chromophore, the prerefined conformations of BSI and Lumi states where the chromophore in the β-ionone ring side underwent large displacements were found by the classical MD simulations. The region around the position which was occupied by the β-ionone ring in BSI and Lumi states (Figure 2) was found to be loosely packed in the Rho dark state on the basis of an experimental study by Katayama et al.83 reporting slow H−D exchange of Thr118 due to transit visit of water molecules into the region. The loose packing and expected high flexibility due to breakage of the α-helical structure around His211 in Helix V introduced by Pro215 in the region observed in the Rho state is suggested to allow for the occupation of the β-ionone ring in the region in the formations of BSI and Lumi states after the large conformational change of the chromophore upon photoisomerization. The structural changes in the β-ionone ring side of the chromophore and the surrounding protein groups in the formation of the Lumi state (Figure 3) well explain biochemical and spectroscopic observations. The planar conformation of the polyene chain resulting from its straightening is consistent with

a

The molecular geometries are the same as the free energetically optimized ones. bValues in parentheses are shifts from the Rho state.

chromophore increased by 0.1 eV even without the external electrostatic field upon the formation of the BSI state. The structural changes of the chromophore, e.g., the torsional changes of the polyene chain and/or changes of the bond alternation associated with the electrostatic interaction with the surrounding environment, therefore gave a non-negligible contribution to the blue shift, although, unfortunately, the complex structural changes hamper the definitive identification of the molecular origins. Lumi States. A classical MD simulation was continued for 200 ns after the MD simulation for the BSI state described above. During the MD simulation, an additional water molecule entered into the binding pocket and occupied a cavity in the vicinity of Glu113 created by the conformational changes upon the isomerization of the chromophore. No other distinct conformational changes were visually seen. We then performed QM/MM RWFE-SCF free energy geometry optimizations from two representative snapshots. Figure 3a depicts the molecular structures of the binding pocket of the two optimized G


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The Journal of Physical Chemistry B the lack of a large distortion of the polyene chain observed by resonance Raman and FTIR experiments.9,11 The approach of the β-ionone ring to Ala168 is consistent with the cross-linking experiment by Borhan et al.,14 as mentioned above. On the other hand, Ahuja et al. concluded from their 2D-NMR measurements that the β-ionone ring does not move toward Helix IV; no NMR cross peaks between the Cβ atom of Cys168 and the C5, C6, or C7 carbon atoms of retinal in Meta II were observed.12 However, the distances between C5−C7 atoms and Cys168 in the present Lumi models are longer than 5.5 Å, which is the expected threshold distance in the NMR experiment, even though the approach of the β-ionone ring to Helix IV took place. The distance between Cys168 and C5− C7 atoms of the retinal may not be a good label to detect the displacement of the β-ionone ring. A domino structural rearrangement which was initiated by the displacement of the β-ionone ring and propagated to Tyr206 through Phe203 led to a complete dissociation of a hydrogen bond of the hydroxyl group of Tyr206, which is consistent with a NMR measurement by Ahuja et al.84 The MD simulation time to generate the prerefined conformations of Lumi intermediate states, 200 ns, is consistent with the experimental evidence (Figure 2), while that of the BSI intermediate is underestimated presumably due to lack of sufficient accuracy of the force fields, especially for the chromophore. The highly accurate QM/MM free energy geometry optimizations where the chromophore and several key groups in the binding pocket were treated at the ab initio QM level of theory were therefore performed to refine the structural models and to characterize the spectroscopic properties. The double computational evaluations of both the vibrational frequency and the electronic photoabsorption energy provided more reliable characterization and assessment of the models. A resonance Raman measurement9 revealed that, in the formation of the BSI state, the strength of the hydrogen bond of the protonated Schiff base with its counterion of Glu113 is unchanged while the absorption maximum is remarkably blue-shifted. The present calculations well reproduced those experimentally observed characteristics and identified the molecular origins underlying the blue shift of the absorption maximum. Furthermore, the present calculations also well reproduced the spectroscopic characteristics of the Lumi state, i.e., the red shift of the absorption maximum from the BSI state and the upshift of the vibrational frequency from the Rho and BSI states,9 and revealed that those spectral shifts were caused by the weakening of the hydrogen bond of the protonated Schiff base with Glu113. The weakening of the hydrogen bond is a functionally critical sign of the photoactivation process where the protonated Schiff base is suggested to point toward Glu181 located in the side opposite to Glu113 in the following formation of the Meta-I intermediate state85 (Figure 2). Despite the high consistency of the present theoretical models of the Lumi state with the experimental spectroscopic evidence, the structures are considerably different from an Xray crystallographic one15 (PDB ID: 2HPY), where large displacement of the β-ionone ring is not observed (Figure 1b). The protonated Schiff base in the X-ray crystallographic structure is also modeled to point toward the cytoplasmic side and undergo a complete dissociation of the hydrogen bond with Glu113 (Figure 6a). The structural features of the X-ray crystallographic model are, however, not in line with the mechanistic understanding of the conformational changes

Figure 6. Comparison of structures of the chromophore, Glu113, and a β-sheet adjacent to the chromophore in the Lumi state. (a) An X-ray crystallographic structure15 (PDB ID: 2HPY). A hydrogen atom of the protonated Schiff base was manually placed at a putative position on a sp2 plane of Cε, Nζ, and C15 atoms. (b) A theoretical model of Lumi(1).

obtained in the present study. If the Nζ−H bond of the protonated Schiff base is isolated without a hydrogen-bond partner, the charge separation of the protonated Schiff base and Glu113 would strongly destabilize the system. Moreover, the vibrational frequency of the Nζ−D stretching mode should be much higher than that observed by FTIR measurement as in halorhodopsin.82 In the case of the X-ray crystallographic model, therefore, it is likely that the protonated Schiff base forms a hydrogen bond with a water molecule, although such a water molecule is not seen in the model. However, any sign of the hydrogen-bonding water molecule was also not observed by a FTIR measurement.9 As mentioned above, the activation process after the formation of the Lumi state involves the orientational change of the Schiff base from the Glu113 side to the Glu181 one, which is expected to be accompanied by entire rotation of the polyene chain to keep an all-trans conformation. The structural evolution of the Schiff base region up to the Lumi state seen in the present theoretical models indicates that the rotation of the polyene chain proceeds in the direction where the protonated Schiff base points toward the extracellular side (Figure 6b), while the rotation in the X-ray crystallographic model15 is suggested to occur in the opposite direction (Figure 6a). In the case of the latter, however, the methyl group attached to the polyene chain at the position of 13 would encounter steric conflict with a β-sheet closely lying next to the polyene chain on the extracellular side during the rotation. On the other hand, the methyl groups can avoid the steric conflict with the β-sheet during the rotation of the polyene chain in the direction in the present theoretical models. Furthermore, the region on the cytoplasmic side of the polyene chain through which the methyl group at the position 13 passes during the rotation in the theoretical models contains the side chain of Trp265, which is expected to be flexible in the Lumi state due to the large displacement of the β-ionone ring originally attached to the side chain of Trp265 in the Rho state (Figures 1b and 3a). The expected direction of the rotation of the polyene chain revealed H


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Innovative Drug Discovery Infrastructure Through Functional Control of Biomolecular Systems)) and the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry. Computations were partly performed at the Research Center for Computational Science, Okazaki, Japan.

by the present theoretical models is therefore suggested to enable more efficient formation of the Meta-I state.

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CONCLUSIONS We performed theoretical modeling of the intermediate states of the photoactivation processes of Rh as well as the dark state by means of ab initio QM/MM free energy geometry optimizations. The geometry refinements along with the evaluations of both vibrational and electronic spectroscopic properties with accurate ab initio QM treatment of the ligand chromophore and the key groups interacting with it and ample statistical samples of the molecular conformations of the surrounding protein environment successfully identified and characterized the molecular events underlying the photoactivation processes. The formation of the BSI intermediate was characterized by the large displacement of the β-ionone ring of the retinal chromophore, which then induced the weakening of the hydrogen bond of the protonated Schiff base with its counterion, Glu113, in the following formation of the Lumi intermediate. The structural evolution revealed in the calculations suggests efficient conformational changes of the chromophore in subsequent activation processes to the Meta-I state. The present theoretical approach with the ab initio QM/ MM free energy geometry optimizations will also be applicable to activation/inactivation processes of other G-protein coupled receptors with complex ligands.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b13050. Decomposed electrostatic energy contributions to excitation energies, electrostatic contributions of water molecules to excitation energies, C−H bond order parameters of POPC lipid molecules in the membrane in the dark state, and QM regions in the QM/MM RWFE-SCF geometry optimizations of Rho, BSI, Lumi(1), and Lumi(2) (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-75-7534006. ORCID

Shigehiko Hayashi: 0000-0003-2658-3171 Present Address †

M.K.: Advanced Institute for Computational Science, RIKEN, 7-6-1 minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. Author Contributions

M.K. performed the calculations. M.K. and S.H. designed the research and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.H. is grateful to the late Klaus Schulten for many things he taught S.H. in life. This work was financially supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology (25104004, 25291034, 16H04776, and “Priority Issue on Post-Kcomputer” (Building I


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Photoactivation Intermediates of a G-Protein Coupled Receptor

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