Activation of the G-Protein-Coupled Receptor Rhodopsin by Water.

Visual rhodopsin is an important archetype for G-protein-coupled receptors, which are membrane proteins implicated in cellular signal transduction. Herein, we show experimentally that approximately 80 water molecules flood rhodopsin upon light absorption to form a solvent-swollen active state. An influx of mobile water is necessary for activating the photoreceptor, and this finding is supported by molecular dynamics (MD) simulations. Combined force-based measurements involving osmotic and hydrostatic pressure indicate the expansion occurs by changes in cavity volumes, together with greater hydration in the active metarhodopsin-II state. Moreover, we discovered that binding and release of the C-terminal helix of transducin is coupled to hydration changes as may occur in visual signal amplification. Hydration-dehydration explains signaling by a dynamic allosteric mechanism, in which the soft membrane matter (lipids and water) has a pivotal role in the catalytic G-protein cycle.

A Usual G-Protein-Coupled Receptor in Unusual Membranes.

G-protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and constitute about 50% of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge-interaction-directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid- and polymer-based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non-biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H(+)] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin-conserved ERY sequence motif that helps break the cytoplasmic "ionic lock". This study unveils a novel design concept of non-biological membranes to reconstitute and harness GPCR functions in synthetic systems.

Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4ß6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1ß1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the ß2-AR*·Gαsß1γ2 complex crystal structure, the Gαi1ß1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1ß1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins.

Global fold of human cannabinoid type 2 receptor probed by solid-state 13C-, 15N-MAS NMR and molecular dynamics simulations.

The global fold of human cannabinoid type 2 (CB2 ) receptor in the agonist-bound active state in lipid bilayers was investigated by solid-state (13)C- and (15)N magic-angle spinning (MAS) NMR, in combination with chemical-shift prediction from a structural model of the receptor obtained by microsecond-long molecular dynamics (MD) simulations. Uniformly (13)C- and (15)N-labeled CB2 receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. (13)C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of Cα, Cß, and C=O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the (13) C chemical shift distribution of Cα resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB2. Thus the shape of the Cα band can be used as an indicator of CB2 global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥ 1.5 ppm for carbons and ≥ 5.0 ppm for nitrogens). Simulated two-dimensional (13) Cα(i)-(13)C=O(i) and (13)C=O(i)-(15)NH(i + 1) dipolar-interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB2 by solid-state MAS NMR.

Retinal ligand mobility explains internal hydration and reconciles active rhodopsin structures.

Rhodopsin, the mammalian dim-light receptor, is one of the best-characterized G-protein-coupled receptors, a pharmaceutically important class of membrane proteins that has garnered a great deal of attention because of the recent availability of structural information. Yet the mechanism of rhodopsin activation is not fully understood. Here, we use microsecond-scale all-atom molecular dynamics simulations, validated by solid-state (2)H nuclear magnetic resonance spectroscopy, to understand the transition between the dark and metarhodopsin I (Meta I) states. Our analysis of these simulations reveals striking differences in ligand flexibility between the two states. Retinal is much more dynamic in Meta I, adopting an elongated conformation similar to that seen in the recent activelike crystal structures. Surprisingly, this elongation corresponds to both a dramatic influx of bulk water into the hydrophobic core of the protein and a concerted transition in the highly conserved Trp265(6.48) residue. In addition, enhanced ligand flexibility upon light activation provides an explanation for the different retinal orientations observed in X-ray crystal structures of active rhodopsin.

Molecular dynamics simulations reveal specific interactions of post-translational palmitoyl modifications with rhodopsin in membranes.

We present a detailed analysis of the behavior of the highly flexible post-translational lipid modifications of rhodopsin from multiple-microsecond all-atom molecular dynamics simulations. Rhodopsin was studied in a realistic membrane environment that includes cholesterol, as well as saturated and polyunsaturated lipids with phosphocholine and phosphoethanolamine headgroups. The simulation reveals striking differences between the palmitoylations at Cys322 and Cys323 as well as between the palmitoyl chains and the neighboring lipids. Notably the palmitoyl group at Cys322 shows considerably greater contact with helix H1 of rhodopsin, yielding frequent chain upturns with longer reorientational correlation times, and relatively low order parameters. While the palmitoylation at Cys323 makes fewer protein contacts and has increased order compared to Cys322, it nevertheless exhibits greater flexibility with smaller order parameters than the stearoyl chains of the surrounding lipids. The dynamical structure of the palmitoylations-as well as their extensive fluctuations-suggests a complex function for the post-translational modifications in rhodopsin and potentially other G protein-coupled receptors, going beyond their role as membrane anchoring elements. Rather, we propose that the palmitoylation at Cys323 has a potential role as a lipid anchor, whereas the palmitoyl-protein interaction observed for Cys322 suggests a more specific interaction that affects the stability of the dark state of rhodopsin.

Dynamics of confined reactive water in smectite clay-zeolite composites.

The dynamics of water confined to mesoporous regions in minerals such as swelling clays and zeolites is fundamental to a wide range of resource management issues impacting many processes on a global scale, including radioactive waste containment, desalination, and enhanced oil recovery. Large-scale atomic models of freely diffusing multilayer smectite particles at low hydration confined in a silicalite cage are used to investigate water dynamics in the composite environment with the ReaxFF reactive force field over a temperature range of 300-647 K. The reactive capability of the force field enabled a range of relevant surface chemistry to emerge, including acid/base equilibria in the interlayer calcium hydrates and silanol formation on the edges of the clay and inner surface of the zeolite housing. After annealing, the resulting clay models exhibit both mono- and bilayer hydration structures. Clay surface hydration redistributed markedly and yielded to silicalite water loading. We find that the absolute rates and temperature dependence of water dynamics compare well to neutron scattering data and pulse field gradient measures from relevant samples of Ca-montmorillonite and silicalite, respectively. Within an atomistic, reactive context, our results distinguish water dynamics in the interlayer Ca(OH)(2)·nH(2)O environment from water flowing over the clay surface, and from water diffusing within silicalite. We find that the diffusion of water when complexed to Ca hydrates is considerably slower than freely diffusing water over the clay surface, and the reduced mobility is well described by a difference in the Arrhenius pre-exponential factor rather than a change in activation energy.

Performance of hybrid programming models for multiscale cardiac simulations: preparing for petascale computation.

Future multiscale and multiphysics models that support research into human disease, translational medical science, and treatment can utilize the power of high-performance computing (HPC) systems. We anticipate that computationally efficient multiscale models will require the use of sophisticated hybrid programming models, mixing distributed message-passing processes [e.g., the message-passing interface (MPI)] with multithreading (e.g., OpenMP, Pthreads). The objective of this study is to compare the performance of such hybrid programming models when applied to the simulation of a realistic physiological multiscale model of the heart. Our results show that the hybrid models perform favorably when compared to an implementation using only the MPI and, furthermore, that OpenMP in combination with the MPI provides a satisfactory compromise between performance and code complexity. Having the ability to use threads within MPI processes enables the sophisticated use of all processor cores for both computation and communication phases. Considering that HPC systems in 2012 will have two orders of magnitude more cores than what was used in this study, we believe that faster than real-time multiscale cardiac simulations can be achieved on these systems.

Orthogonal recursive bisection as data decomposition strategy for massively parallel cardiac simulations.

We present the orthogonal recursive bisection algorithm that hierarchically segments the anatomical model structure into subvolumes that are distributed to cores. The anatomy is derived from the Visible Human Project, with electrophysiology based on the FitzHugh-Nagumo (FHN) and ten Tusscher (TT04) models with monodomain diffusion. Benchmark simulations with up to 16,384 and 32,768 cores on IBM Blue Gene/P and L supercomputers for both FHN and TT04 results show good load balancing with almost perfect speedup factors that are close to linear with the number of cores. Hence, strong scaling is demonstrated. With 32,768 cores, a 1000 ms simulation of full heart beat requires about 6.5 min of wall clock time for a simulation of the FHN model. For the largest machine partitions, the simulations execute at a rate of 0.548 s (BG/P) and 0.394 s (BG/L) of wall clock time per 1 ms of simulation time. To our knowledge, these simulations show strong scaling to substantially higher numbers of cores than reported previously for organ-level simulation of the heart, thus significantly reducing run times. The ability to reduce runtimes could play a critical role in enabling wider use of cardiac models in research and clinical applications.

Petascale computation performance of lightweight multiscale cardiac models using hybrid programming models.

Future multiscale and multiphysics models must use the power of high performance computing (HPC) systems to enable research into human disease, translational medical science, and treatment. Previously we showed that computationally efficient multiscale models will require the use of sophisticated hybrid programming models, mixing distributed message passing processes (e.g. the message passing interface (MPI)) with multithreading (e.g. OpenMP, POSIX pthreads). The objective of this work is to compare the performance of such hybrid programming models when applied to the simulation of a lightweight multiscale cardiac model. Our results show that the hybrid models do not perform favourably when compared to an implementation using only MPI which is in contrast to our results using complex physiological models. Thus, with regards to lightweight multiscale cardiac models, the user may not need to increase programming complexity by using a hybrid programming approach. However, considering that model complexity will increase as well as the HPC system size in both node count and number of cores per node, it is still foreseeable that we will achieve faster than real time multiscale cardiac simulations on these systems using hybrid programming models.

A lipid pathway for ligand binding is necessary for a cannabinoid G protein-coupled receptor.

Recent isothiocyanate covalent labeling studies have suggested that a classical cannabinoid, (-)-7'-isothiocyanato-11-hydroxy-1',1'dimethylheptyl-hexahydrocannabinol (AM841), enters the cannabinoid CB2 receptor via the lipid bilayer (Pei, Y., Mercier, R. W., Anday, J. K., Thakur, G. A., Zvonok, A. M., Hurst, D., Reggio, P. H., Janero, D. R., and Makriyannis, A. (2008) Chem. Biol. 15, 1207-1219). However, the sequence of steps involved in such a lipid pathway entry has not yet been elucidated. Here, we test the hypothesis that the endogenous cannabinoid sn-2-arachidonoylglycerol (2-AG) attains access to the CB2 receptor via the lipid bilayer. To this end, we have employed microsecond time scale all-atom molecular dynamics (MD) simulations of the interaction of 2-AG with CB2 via a palmitoyl-oleoyl-phosphatidylcholine lipid bilayer. Results suggest the following: 1) 2-AG first partitions out of bulk lipid at the transmembrane alpha-helix (TMH) 6/7 interface; 2) 2-AG then enters the CB2 receptor binding pocket by passing between TMH6 and TMH7; 3) the entrance of the 2-AG headgroup into the CB2 binding pocket is sufficient to trigger breaking of the intracellular TMH3/6 ionic lock and the movement of the TMH6 intracellular end away from TMH3; and 4) subsequent to protonation at D3.49/D6.30, further 2-AG entry into the ligand binding pocket results in both a W6.48 toggle switch change and a large influx of water. To our knowledge, this is the first demonstration via unbiased molecular dynamics that a ligand can access the binding pocket of a class A G protein-coupled receptor via the lipid bilayer and the first demonstration via molecular dynamics of G protein-coupled receptor activation triggered by a ligand binding event.

Concerted interconversion between ionic lock substates of the beta(2) adrenergic receptor revealed by microsecond timescale molecular dynamics.

The recently solved crystallographic structures for the A(2A) adenosine receptor and the beta(1) and beta(2) adrenergic receptors have shown important differences between members of the class-A G-protein-coupled receptors and their archetypal model, rhodopsin, such as the apparent breaking of the ionic lock that stabilizes the inactive structure. Here, we characterize a 1.02 mus all-atom simulation of an apo-beta(2) adrenergic receptor that is missing the third intracellular loop to better understand the inactive structure. Although we find that the structure is remarkably rigid, there is a rapid influx of water into the core of the protein, as well as a slight expansion of the molecule relative to the crystal structure. In contrast to the x-ray crystal structures, the ionic lock rapidly reforms, although we see an activation-precursor-like event wherein the ionic lock opens for approximately 200 ns, accompanied by movements in the transmembrane helices associated with activation. When the lock reforms, we see the structure return to its inactive conformation. We also find that the ionic lock exists in three states: closed (or locked), semi-open with a bridging water molecule, and open. The interconversion of these states involves the concerted motion of the entire protein. We characterize these states and the concerted motion underlying their interconversion. These findings may help elucidate the connection between key local events and the associated global structural changes during activation.

Strong scaling and speedup to 16,384 processors in cardiac electro-mechanical simulations.

High performance computing is required to make feasible simulations of whole organ models of the heart with biophysically detailed cellular models in a clinical setting. Increasing model detail by simulating electrophysiology and mechanical models increases computation demands. We present scaling results of an electro - mechanical cardiac model of two ventricles and compare them to our previously published results using an electrophysiological model only. The anatomical data-set was given by both ventricles of the Visible Female data-set in a 0.2 mm resolution. Fiber orientation was included. Data decomposition for the distribution onto the distributed memory system was carried out by orthogonal recursive bisection. Load weight ratios for non-tissue vs. tissue elements used in the data decomposition were 1:1, 1:2, 1:5, 1:10, 1:25, 1:38.85, 1:50 and 1:100. The ten Tusscher et al. (2004) electrophysiological cell model was used and the Rice et al. (1999) model for the computation of the calcium transient dependent force. Scaling results for 512, 1024, 2048, 4096, 8192 and 16,384 processors were obtained for 1 ms simulation time. The simulations were carried out on an IBM Blue Gene/L supercomputer. The results show linear scaling from 512 to 16,384 processors with speedup factors between 1.82 and 2.14 between partitions. The most optimal load ratio was 1:25 for on all partitions. However, a shift towards load ratios with higher weight for the tissue elements can be recognized as can be expected when adding computational complexity to the model while keeping the same communication setup. This work demonstrates that it is potentially possible to run simulations of 0.5 s using the presented electro-mechanical cardiac model within 1.5 hours.

Orthogonal recursive bisection data decomposition for high performance computing in cardiac model simulations: dependence on anatomical geometry.

Orthogonal recursive bisection (ORB) algorithm can be used as data decomposition strategy to distribute a large data set of a cardiac model to a distributed memory supercomputer. It has been shown previously that good scaling results can be achieved using the ORB algorithm for data decomposition. However, the ORB algorithm depends on the distribution of computational load of each element in the data set. In this work we investigated the dependence of data decomposition and load balancing on different rotations of the anatomical data set to achieve optimization in load balancing. The anatomical data set was given by both ventricles of the Visible Female data set in a 0.2 mm resolution. Fiber orientation was included. The data set was rotated by 90 degrees around x, y and z axis, respectively. By either translating or by simply taking the magnitude of the resulting negative coordinates we were able to create 14 data set of the same anatomy with different orientation and position in the overall volume. Computation load ratios for non - tissue vs. tissue elements used in the data decomposition were 1:1, 1:2, 1:5, 1:10, 1:25, 1:38.85, 1:50 and 1:100 to investigate the effect of different load ratios on the data decomposition. The ten Tusscher et al. (2004) electrophysiological cell model was used in monodomain simulations of 1 ms simulation time to compare performance using the different data sets and orientations. The simulations were carried out for load ratio 1:10, 1:25 and 1:38.85 on a 512 processor partition of the IBM Blue Gene/L supercomputer. Th results show that the data decomposition does depend on the orientation and position of the anatomy in the global volume. The difference in total run time between the data sets is 10 s for a simulation time of 1 ms. This yields a difference of about 28 h for a simulation of 10 s simulation time. However, given larger processor partitions, the difference in run time decreases and becomes less significant. Depending on the processor partition size, future work will have to consider the orientation of the anatomy in the global volume for longer simulation runs.

Structural and dynamic effects of cholesterol at preferred sites of interaction with rhodopsin identified from microsecond length molecular dynamics simulations.

An unresolved question about GPCR function is the role of membrane components in receptor stability and activation. In particular, cholesterol is known to affect the function of membrane proteins, but the details of its effect on GPCRs are still elusive. Here, we describe how cholesterol modulates the behavior of the TM1-TM2-TM7-helix 8(H8) functional network that comprises the highly conserved NPxxY(x)(5,6)F motif, through specific interactions with the receptor. The inferences are based on the analysis of microsecond length molecular dynamics (MD) simulations of rhodopsin in an explicit membrane environment. Three regions on the rhodopsin exhibit the highest cholesterol density throughout the trajectory: the extracellular end of TM7, a location resembling the high-density sterol area from the electron microscopy data; the intracellular parts of TM1, TM2, and TM4, a region suggested as the cholesterol binding site in the recent X-ray crystallography data on beta(2)-adrenergic GPCR; and the intracellular ends of TM2-TM3, a location that was categorized as the high cholesterol density area in multiple independent 100 ns MD simulations of the same system. We found that cholesterol primarily affects specific local perturbations of the helical TM domains such as the kinks in TM1, TM2, and TM7. These local distortions, in turn, relate to rigid-body motions of the TMs in the TM1-TM2-TM7-H8 bundle. The specificity of the effects stems from the nonuniform distribution of cholesterol around the protein. Through correlation analysis we connect local effects of cholesterol on structural perturbations with a regulatory role of cholesterol in the structural rearrangements involved in GPCR function.

Internal hydration increases during activation of the G-protein-coupled receptor rhodopsin.

Rhodopsin, the membrane protein responsible for dim-light vision, until recently was the only G-protein-coupled receptor (GPCR) with a known crystal structure. As a result, there is enormous interest in studying its structure, dynamics, and function. Here we report the results of three all-atom molecular dynamics simulations, each at least 1.5 micros, which predict that substantial changes in internal hydration play a functional role in rhodopsin activation. We confirm with (1)H magic angle spinning NMR that the increased hydration is specific to the metarhodopsin-I intermediate. The internal water molecules interact with several conserved residues, suggesting that changes in internal hydration may be important during the activation of other GPCRs. The results serve to illustrate the synergism of long-time-scale molecular dynamics simulations and NMR in enhancing our understanding of GPCR function.

Large scale cardiac modeling on the Blue Gene supercomputer.

Multi-scale, multi-physical heart models have not yet been able to include a high degree of accuracy and resolution with respect to model detail and spatial resolution due to computational limitations of current systems. We propose a framework to compute large scale cardiac models. Decomposition of anatomical data in segments to be distributed on a parallel computer is carried out by optimal recursive bisection (ORB). The algorithm takes into account a computational load parameter which has to be adjusted according to the cell models used. The diffusion term is realized by the monodomain equations. The anatomical data-set was given by both ventricles of the Visible Female data-set in a 0.2 mm resolution. Heterogeneous anisotropy was included in the computation. Model weights as input for the decomposition and load balancing were set to (a) 1 for tissue and 0 for non-tissue elements; (b) 10 for tissue and 1 for non-tissue elements. Scaling results for 512, 1024, 2048, 4096 and 8192 computational nodes were obtained for 10 ms simulation time. The simulations were carried out on an IBM Blue Gene/L parallel computer. A 1 s simulation was then carried out on 2048 nodes for the optimal model load. Load balances did not differ significantly across computational nodes even if the number of data elements distributed to each node differed greatly. Since the ORB algorithm did not take into account computational load due to communication cycles, the speedup is close to optimal for the computation time but not optimal overall due to the communication overhead. However, the simulation times were reduced form 87 minutes on 512 to 11 minutes on 8192 nodes. This work demonstrates that it is possible to run simulations of the presented detailed cardiac model within hours for the simulation of a heart beat.

Dynamic structure of retinylidene ligand of rhodopsin probed by molecular simulations.

Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the beta-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the beta-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The beta-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state (2)H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.

Convergence of molecular dynamics simulations of membrane proteins.

The central question in evaluating almost any result from a molecular dynamics simulation is whether the calculation has converged. Unfortunately, assessing the ergodicity of a single trajectory is very difficult to do. In this work, we assess the sampling of molecular dynamics simulations of the membrane protein rhodopsin by comparing the results from 26 independent trajectories, each run for 100 ns. By examining principal components and cluster populations, we show that rhodopsin's fluctuations are not well described by 100 ns of dynamics, and that the sampling is not fully converged even for individual loops. The results serve as a reminder of the caution required when interpreting molecular dynamics simulations of macromolecules.

Retinal counterion switch mechanism in vision evaluated by molecular simulations.

Photoisomerization of the retinylidene chromophore of rhodopsin is the starting point in the vision cascade. A counterion switch mechanism that stabilizes the retinal protonated Schiff base (PSB) has been proposed to be an essential step in rhodopsin activation. On the basis of vibrational and UV-visible spectroscopy, two counterion switch models have emerged. In the first model, the PSB is stabilized by Glu181 in the meta I state, while in the most recent proposal, it is stabilized by Glu113 as well as Glu181. We assess these models by conducting a pair of microsecond scale, all-atom molecular dynamics simulations of rhodopsin embedded in a 99-lipid bilayer of SDPC, SDPE, and cholesterol (2:2:1 ratio) varying the starting protonation state of Glu181. Theoretical simulations gave different orientations of retinal for the two counterion switch mechanisms, which were used to simulate experimental 2H NMR spectra for the C5, C9, and C13 methyl groups. Comparison of the simulated 2H NMR spectra with experimental data supports the complex-counterion mechanism. Hence, our results indicate that Glu113 and Glu181 stabilize the retinal PSB in the meta I state prior to activation of rhodopsin.