Selective Protonation of Acidic Residues ... - ACS Publications

Agustín Bruzzese , Carles Gil , James A. R. Dalton , Jesús Giraldo ... Carlos Gueto-Tettay , Alejandro Martinez-Consuegra , Luis Pelaez-Bedoya , Jua...
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The Journal of Physical Chemistry

Selective Protonation of Acidic Residues Triggers Opsin Activation

Isaias Lans, James A. R. Dalton and Jesús Giraldo*

Laboratory of Molecular Neuropharmacology and Bioinformatics, Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain

*Corresponding author: Jesús Giraldo Laboratory of Molecular Neuropharmacology and Bioinformatics, Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona. 08193 Bellaterra, Spain Phone: +34 93 581 38 13 ; Fax : +34 93 581 23 44 ; Email : [email protected]

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ABSTRACT Rhodopsin, the visual pigment in the retina, is a Class A G protein-coupled receptor (GPCR) covalently bound to retinal chromophore. In dark conditions, retinal is in the cis-isomeric state, stabilizing the rhodopsin inactive state as an inverse agonist. After light absorption, retinal undergoes an isomerization photoreaction to trans-retinal, which includes a conformational change of the receptor to its active state. In absence of retinal, the apoprotein opsin presents a low level of constitutive activity, which depends on pH and with higher propensity of activation at acidic pH. To examine the effect and the underlying mechanism that protonation may have on opsin activation, a number of MD simulations were run varying the number and identity of acidic residues selected for protonation. Results show that the combined protonation of D83, E113 and E247 is of special relevance for the induction of receptor activation. Subsequent conformational analysis of the MD trajectories provides a structural mechanistic insight into opsin activation process. Furthermore, because protonation seems to be a determining step in the activation of other GPCRs, the methodology and rationale used herein can be extended to mechanistic studies of GPCRs in general.

Keywords: rhodopsin; opsin; GPCR; receptor activation; protonation; pH; molecular dynamics .

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INTRODUCTION G protein-coupled receptors (GPCRs) are transmembrane proteins responsible for the transduction of signals from the extracellular space to the cytoplasm.1,2 GPCRs mediate a large number of cellular responses to external stimuli both chemical (hormones and neurotransmitters) and physical (light) and, as a consequence, they are associated with numerous diseases. Because of this, GPCRs occupy a central position in the drug pipeline of the pharmaceutical industry.3 In this regard, about one third of all drugs that were approved by the US Food and Drug Administration during the past three decades target GPCRs.4 GPCRs are structurally characterized by seven transmembrane hydrophobic helices (TM1 to TM7) and one intracellular amphipathic helix (H8). The TM helices are connected by three intracellular and three extracellular loops, which in some cases, show secondary structure. GPCRs can be classified into four main classes: class A rhodopsin-like, class B secretin- and adhesion-like, class C metabotropic glutamate, and class F frizzled, smoothened and taste2 receptors.5 In the process of signal transduction GPCRs undergo a conformational change from their inactive to their active state. In the active state GPCRs are able to couple an effector molecule such as the heterotrimeric G protein which, after activation, initiates a spectrum of pleiotropic changes in many targets. The active state of the receptor is stabilized by agonists. The binding of agonists to the extracellular end of GPCRs leads to large structural rearrangements in the cytoplasmic side of the transmembrane domains of the receptors in such a way that it facilitates the binding of the G protein. In contrast, the binding of inverse agonists leads to the stabilization of the inactive state, which hampers G protein binding.6 Crystallographic structures of 22 class A GPCRs have been solved by the date on which this article was submitted (Ref. 7 as of February

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2015) in different states, inactive with inverse agonists bound, intermediately active with agonists bound and the fully active state, in which the receptor is bound to both an agonist and the G protein (see Refs. 1,2,5,8 for review). Less is known with respect to the structures of the other GPCR families. Recently, one class F,9 two class B10,11 and the TM domains of two metabotropic glutamate class C12,13 receptors, all in their inactive state, have been solved. Several experimental and computational studies have investigated the GPCR activation process14,15,16,17,18. The largest changes are observed in the cytoplasmic side of the receptor structures, where the intracellular end of TM6 points outward from the transmembrane core in the active structures and the intermediate active states show TM6 conformations between active and inactive states. This movement of TM6, which seems to occur in a concerted fashion with TM5, appears to be conserved in all class A GPCRs and is fundamental for G protein binding and activation.19 In contrast, less pronounced movements involving TM3 and TM7 seem to depend on particular receptor and ligand combinations and might contribute to G protein–independent signaling pathways.1 The movements of the TM helices in the receptor activation process are possible thanks to the intervention of a number of molecular microswitches along the TM domain.1,20,21,22 These microswitches include: (i) the ionic lock switch, a salt bridge between R3.50 and E6.30 residues (named according to Ballesteros-Weinstein numbering system)23 at the intracellular ends of TM3 and TM6, which is characteristic of inactive states; (ii) the 3-7 lock switch, an interaction involving E3.28 and K7.43 residues in rhodopsin and other acidic residues and positions (D3.32) in other GPCRs; (iii) the transmission switch, also known as the rotamer toggle switch, a molecular switch that is comprised of the conserved residues W6.48 and F6.44, which show a conformational change in receptor activation; and (iv) the tyrosine microswitches, two tyrosine

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residues, Y7.53 (92% conserved) and Y5.58 (89% conserved), involved in GPCR activation in connection with the so called hydrophobic barrier and a water-mediated hydrogen bond network. The present study focuses on the activation of the class A GPCR rhodopsin, a pigment covalently bound to retinal chromophore by the formation of a protonated Schiff base (PSB) via K2967.43. In dark conditions, retinal is in the cis-isomeric state, stabilizing the rhodopsin inactive state as an inverse agonist. After light absorption, retinal undergoes an isomerization photoreaction to trans-retinal, which includes a conformational change of the receptor to its active state.24 Rhodopsin crystal structures include a number of structural and functional features that make this receptor a suitable model for GPCR activation studies.25 Rhodopsin is the only GPCR for which crystal structures both active and inactive ligand-bound and ligand-free are known.24 In addition, and importantly, the presence of the third intracellular loop in rhodopsin structures, which is absent in most structures of other GPCRs, allows for more reliable structure-function analyses.25 In absence of retinal, the apoprotein opsin presents a low level of constitutive activity, which can be increased after M6.40 mutation.26 Most of the mutations on this residue yield opsins with significant constitutive activity indicating the importance of M6.40 in maintaining interhelical TM6-TM7 interactions that constrain receptor activation.19 Protonation of particular residues may produce receptor active structures as suggested from a crystal structure of opsin which is in an active state at the acidic pH of 5.6.27 This is consistent with other GPCRs for which a protonation step for activation has been proposed,28,29 and an indication that, despite the specific properties of rhodopsin associated to covalent ligand binding and photochemical activation, many mechanistic insights from this receptor can be generalized for GPCRs.24

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In the present study we aim at examining the GPCR activation mechanism by taking opsin as a GPCR model, and exploring opsin activation by molecular dynamics (MD) simulations of molecular systems with different combinations of protonation states for the receptor. Our rationale has a remarkable corollary, the proposal to systematically investigate by computational methods potential mechanistic routes that GPCRs in general, and rhodopsin in particular, may follow to trigger transition from inactive to active states. It is also worth mentioning that although activation mechanisms of GPCRs have been investigated by MD before, such as the transition of the β2-adrenergic receptor,14,17 these kinds of unbiased simulations have typically needed to be long (occurring over many microseconds) and ideally performed on specialised computational hardware. On the contrary, here we explore activation pathways of opsin through much shorter unbiased MD simulations occurring over readily accessible time-scales (i.e. nanoseconds) on a regular workstation computer. This is made possible by including the protonation of specific acidic residues.

MATERIALS AND METHODS To study the activation of opsin, we carried out several MD simulations of this receptor in different protonation states in a model membrane environment. The GROMOS53A6 force field30,31 was used. The initial structure was the inactive X-ray rhodopsin structure (PDB code 1GZM),32 in which cis-retinal was removed to obtain an inactive opsin. We performed all simulations on a standard workstation using GROMACS v4.6.1.30

Minimization The ligand-free crystallographic structure was inserted into a pre-equilibrated and fully hydrated POPC lipid bilayer of 128 molecules. The lipid bilayer was constructed and

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tailored

to

the

intended

size

using

Berger

lipids33

(obtained

from

http://moose.bio.ucalgary.ca/). The embedding protocol was achieved using the oriented structures from OPM database and the inflateGRO methodology.34 The water model used was the single point charge (SPC)65 model. To remove the net charge of the system, Cl- or Na+ ions were added as counterions. Thus, as an example, one Na+ ion was added to the opsin system in which all the acidic residues of the protein were ionized and four Cl- ions were added to the opsin system including five acidic residues in their protonated state. The resulting systems were minimized until the maximum force < 1000.0 kJ/mol/nm.

Equilibration and MD production The equilibration was carried out in three steps. The first step consisted of 100 ps of NVT simulation, in which the z-coordinate of the POPC phosphorous atoms in the membrane as well as the heavy atoms of transmembrane domain were restrained. The second step consisted of 2 substeps, each of them of 2 ns of NPT simulation (2 ns restraining both the z-coordinate of the POPC phosphorous atoms in the membrane and the heavy atoms of the transmembrane domain and 2 ns restraining only the heavy atoms of the transmembrane domain). Finally, the third step consisted of 2 ns without any restraints. MD simulation production runs ranging between 52 ns and 156 ns were then carried out without bias. Furthermore, to provide more conformational sampling and more confidence in the observed results, two longer (260 ns) MD simulations were performed: one including the collection of protonated acidic residues that best promotes opsin activation and the other with all the acidic residues in their ionized state, for control purposes.

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Potential of mean forces calculation The Potential of Mean Force (PMF) was calculated from the MD production phase using the collections of structures saved every 2 ps. The probability density of configurations along the reaction coordinate was collected and sorted into bins of width 0.02 nm (one PMF was repeated using a width of 0.01 to assess that bin size does not bias the results). The reaction coordinates used were: (i) the distance (d1) between the distal carbon atoms Cξ of R1353.50 in TM3 and Cδ of E2476.30 in TM6 (these residues define the ionic lock; d1 = 0.48 nm and 1.79 nm in the inactive 1GZM32 and active 3PQR35 rhodopsin crystal structures, respectively); and (ii) the distance (d2) between the Cδ of E2496.32 in TM6 and the Cα of N3108.47 in the TM7–H8 junction (d2 = 0.40 nm and 0.88 nm in the inactive 1GZM and active 3PQR rhodopsin structures, respectively). These two distances (d1, d2) measure the differences in TM6 between active and inactive crystal structures and may be used as conformational indicators of the outward movement of TM6 in the activation process. Thus, while d1 measures the displacement of TM6 from TM3 on rhodopsin activation, d2 helps to better define the active state by restricting the conformational space. We assumed that both distances need to be significantly greater in an active state than those in the inactive crystal structures in order to consider a receptor conformation as a putative active state.

RESULTS AND DISCUSSION Several studies on class A GPCRs have identified the relationship between the protonation state of some residues and the receptor activation/deactivation process.17,36,37 Interestingly, there exists an X-ray structure of opsin obtained at low pH which is in an active state, suggesting that protonation of particular acidic residues may play a role in receptor activation.27 Additionally, since the highest contributions to the

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stabilization of receptor inactive state arise from ionic interactions, protonation of some of the residues involved may release the constraining effects of these binding forces. Based on these data we ran several MD simulations (along nanosecond time-scales) of opsin in which different combinations of protonation states for D832.50, E1133.28, E1343.49, E2476.30 and E2496.32 were considered. This allowed us to examine potential receptor activation mechanisms in terms of particular titratable residues and provide new insights into the way in which retinal isomerization transmits the activation message from the ligand binding site to the intracellular side. Simulations started with two protonated acidic residues and this number was successively increased up to five.

Activation of opsin by successive residue protonation Two protonated residues D832.50 and E1133.28 were selected for the mechanistic proposal of two protonated acidic residues because of the following reasons: In the case of D83, both experimental spectroscopic data38 and calculated pKa suggest that this residue is protonated at physiological pH (pKa values of 9.37 and 8.76 were obtained for D83 in inactive 1GZM and active 3PQR (PDB codes) rhodopsin crystal structures, respectively, by using the PROPKA program).39 In the case of E1133.28, protonation of this residue has been proposed as the first activation switch in rhodopsin.38 In the inactive state, 11-cis retinal is covalently linked to K2967.43 via a PSB, with E1133.28 acting as a counterion. After light absorption by retinal, and its subsequent conformational isomerization to all-trans, there is a proton transfer from PSB to E1133.28,40 thus facilitating the breaking of the E1133.28-K2967.43 ionic interaction.

→ Figure 1

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Figure 1 displays a scatter plot of the selected (d1, d2) interhelical distances (see Methods) from our MD simulation with both D832.50 and E1133.28 protonated (the protonated 83-113 state). Conformations with either large d1, indicating a separation between R1353.50 and E2476.30, or large d2, indicating a separation between E2496.32 and N3108.47, were found but not with both distances being simultaneously large, indicating that an active conformation was not reached. In fact, the point on the plot bearing the coordinates of the active 3PQR crystal structure (green square) is far away from the sampled space. In addition, the very low value of the calculated correlation coefficient between d1 and d2 (R = -0.143) suggests that there is not an efficient coupling of the corresponding movements between helices.

Three protonated residues The above results indicate that the protonation of two residues, D832.50 and E1133.28, is not enough for the activation of opsin. Thus, we decided to include a third acidic residue in a protonated state. First, we carried out a MD simulation with D832.50, E1133.28 and E1343.49 protonated (the protonated 83-113-134 state). Protonation of E1343.49 from the solvent has been reported as the second activation switch in rhodopsin, and a prerequisite for complete receptor activation under physiological conditions.38 Figure 2A displays a scatter plot of the selected (d1, d2) interhelical distances (see Methods) for the MD simulation corresponding to the protonated 83-113-134 state. Visual comparison between Figures 1 and 2A shows that the inclusion of protonated E1343.49 increases the trend of opsin to adopt active conformations, mainly in terms of d1 distance; however, the reference point (d1=1.79 nm, d2=0.88 nm; green square on the plot), which corresponds to the active 3PQR rhodopsin structure, is not included in the

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sampled space, which indicates that conformations are still far from being active. Second, we carried out a MD simulation with D832.50, E1133.28 and E2496.32 protonated (the protonated 83-113-249 state). Inclusion of protonated E2496.32 was based on the interaction, present in inactive rhodopsin structures, between this residue and N3108.47 in TM7–H8 junction, which constitutes a molecular anchor for TM6 conformational movements. The results (Figure 2B) were similar to the previous simulation. Finally, we carried out a MD simulation with D832.50, E1133.28 and E2476.30 protonated (the protonated 83-113-247 state). Inclusion of protonated E2476.30 was based on this residue being part of the R1353.50-E2476.30 ionic lock, an interaction commonly associated with inactive states. The results (Figure 2C) showed a significant increase in the trend of opsin for the conformational transition to active structures (the point on the plot bearing the coordinates of the active 3PQR crystal structure (green square) is close to the boundaries of the sampled space), thus suggesting that the local pH and therefore the protonation state of particular acidic residues are key for the activation process. In this regard, it has been shown by using Fourier transform infrared spectroscopy experiments, in which E2476.30 and E2496.32 were mutated to glutamine, that neutralization of these glutamate residues plays a role during rhodopsin activation.41 Comparison between Figures 2C and both 2A and 2B shows that not only are higher values of d1 and d2 found in Figure 2C but also an ellipsoid form of the scatter plot, which suggest a coupling movement between the involved helices. This is quantitatively confirmed by the correlation coefficient between d1 and d2, which is 0.833 for Figure 2C in comparison with the much lower values of 0.109 and 0.107 for Figures 2A and 2B, respectively. To verify the hypothesized correspondence between d1 and d2 values in our simulations and receptor activation, Figure 2D shows a structural snapshot of a minimum of a PMF calculation generated from the MD trajectory corresponding to

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Figure 2C (see below on how this minimum is obtained). The calculated structure, which features d1= 1.39 nm and d2=0.88 nm values shows a high resemblance with active rhodopsin crystal structure.

→ Figure 2

Four protonated residues A MD simulation was carried out including D832.50, E1133.28, E2476.30 and E2496.32 in their protonated form (the protonated 83-113-247-249 state). Of note, the simulation includes relatively high values for d1, indicating the breaking of the R1353.50-E2476.30 ionic lock (Figure 3A). However, high d1 values are not accompanied by high d2 (R=0.081) and no active state was generated. A second simulation involving the four protonated residues D832.50, E1133.28, E1343.49 and E2476.30 was carried out (the protonated 83-113-134-247 state). Interestingly, the simulation shows a separation between E2496.32 and N3108.47 (higher d2 distance) for some of the structures (Figure 3B); yet this does not occur as d1 increases (R = -0.052) and, consequently, here again receptor activation is not obtained.

→ Figure 3

Five protonated residues We have shown above that the combined protonation of three particular acidic residues, D832.50, E1133.28 and E2476.30 (the protonated 83-113-247 state), allowed the generation of opsin conformations close to the active state in relatively short MD simulations. However, protonation of a fourth acidic residue, either E134 or E249, did not increase

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the efficiency of the activation process in our simulations. On the contrary, in the protonated 83-113-247-249 state (Figure 3A), the active state is less visited than in the protonated 83-113-247 state (Figure 2C) and in the protonated 83-113-134-247 state (Figure 3B) the capability of opsin for activation was even more limited. Notably, the simultaneous protonation of D832.50, E1133.28, E1343.49, E2476.30 and E2496.32 (the protonated 83-113-134-247-249 state) produced a number of conformations that can be considered to be close to the active state (Figures 4A and 4B). Similar to what happened with the protonated 83-113-247 state, a relatively high correlation coefficient (R = 0.710) was obtained, suggesting again that receptor activation involves the coupling of helix movements involving d1 and d2. Thus, we see that the protonation effect is not additive: while the addition of either E1343.49 or E2496.32 protonated residues to the protonated 83-113-247 state hampers the induction of the active state, the simultaneous protonation of both residues to obtain a five protonated state (the protonated 83-113134-247-249 state) is more prone to the in silico activation of opsin. Nevertheless, comparison of the sampled conformational spaces of 83-113-247 and 83-113-134-247249 states in comparison with the coordinates of the active 3PQR rhodopsin crystal structure (Figures 2C and 4A) indicates that the former protonation switch has higher capability for receptor activation than the latter and that, consequently, receptor activation is not achieved by merely increasing the number of protonated acidic residues.

→ Figure 4

Protonation of E2476.30 is important for opsin activation but not enough if only two acidic residues are protonated

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The addition of a protonated E2476.30 to the combination of protonated D832.50 and E1133.28 residues allowed us to reach conformations resembling active opsin. This result suggests that protonation of E2476.30 is of particular importance in the activation process. Thus, we decided to tackle two separate combinations of pairs of protonated residues: D832.50 and E2476.30 (Supplementary Information. Figure S1A) and E1133.28 and E2476.30 (Supplementary Information. Figure S1B). We see that breaking of the R1353.50-E2476.30 interaction is easier in the former case whereas some additional conformations corresponding to lower E2496.32-N3108.47 interactions appear in the latter case. However, in both protonated states we are far from obtaining active opsin conformations. As expected, low correlation coefficients are obtained in both cases: R = 0.266 and R = 0.001 for 83-247 and 113-247 protonated states, respectively. These results suggest that at least three residues need to be protonated for opsin activation with E2476.30 being of particular relevance.

Computational assessment of the proposed protonic activation switch To further assess the 83-113-247 three-protonated residue state as the simplest combination of protonated residues able to promote opsin activation, we extended the original MD production run from 54 ns to 260 ns. In addition and for control purposes, a new MD simulation with the same production run (260 ns) and with all the acidic residues in their ionized state was performed. Figures 5A and 5B show the scatter plots obtained from the 260 ns MD trajectories of both protonated and ionized states, respectively. The shape of the scatter plot of the 83-113-247 three-protonated residue state in the extended dynamics (Figure 5A) is similar to that of the shorter dynamics (Figure 2C) though including points with higher d1 and d2 values. Importantly, the (d1, d2) point corresponding to the active rhodopsin crystal structure which was close to the

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boundaries of the sampled region in the shorter MD simulation is clearly within the sampled region in the longer MD. On the contrary, the scatter plot of the control simulation displays a very different shape (Figure 5B). The ellipsoid form obtained in the proposed protonic switch, both in protonated 83-113-247 (Figures 2C and 5A) and 83-113-134-247-249 (Figure 4A) states, is not found in the unprotonated state, for which a much lower correlation coefficient is calculated. Moreover, as shown in these figures the maximum d1 and d2 values from the control simulation are clearly below those from simulations with three or five protonated acidic residues, indicating the low capability of the all-ionized acidic residues state for activating the receptor. Finally, Figures 5C and 5D show structural snapshots of minima obtained from PMF calculations generated from the MD trajectories corresponding to Figures 5A and 5B, respectively. In Supplementary Information, Figures S4 to S11 show the PMF plots for these MD simulations and those described above. The presence and location in the plots of the minimum corresponding to an active-like state clearly distinguish the 83-113-247 state from the others. The calculated active and inactive structures (Figures 5C and 5D, respectively), which feature (d1, d2) pairs: (1.35, 0.88) and (1.07, 0.44), respectively, show a high resemblance with 3PQR active and 1GZM inactive rhodopsin crystal structures, respectively.

→ Figure 5

Structural characterization of computational active-like states To structurally characterize those conformations in our titration scheme that most resemble intermediate or active states, a PMF was calculated from the MD simulations corresponding to both the protonated 83-113-247 (scatter plot in Figure 2C) and the

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protonated 83-113-134-247-249 states (scatter plot in Figure 4A). The d1 distance between the residues constituting the ionic lock, as defined in Methods, was taken as the reaction coordinate. For both combinations of protonated residues minima corresponding to intermediate states were obtained (Figure 6A). Comparing the minima with greatest d1 between the curves from three and five protonated residues, we see that the minimum corresponding to the 83-113-247 state is closer to an active state structure whereas that of the 83-113-134-247-249 state is broader and seems to include two similar structures. A second PMF was performed from the same MD simulations by using the distance d2 between E2496.32 and N3108.47, as defined in Methods. Similarly localized minima for the intermediate conformations were obtained for both protonation states (Figure 6B). From the minima in Figures 6A and 6B the collection of structures comprised in the two PMF window spaces (both d1 and d2) including the minima nearest to the active state were selected. To study in more detail their structure-function characteristics we examined these structures in terms of the molecular microswitches commonly discussed in the literature1,20,21,22 (see microswitches i to iv in the Introduction). The sampled structures were those corresponding to the intersection between the two minima involving d1 and d2 distances nearest to the active states. Table 1 shows the distributions of sample values (n, mean ± SEM) in comparison with inactive and active rhodopsin crystal structures 1GZM and 3PQR, respectively, for: (i) The ionic lock switch (R3.50 and E6.30): Distance between Cξ of R3.50 and Cδ of E6.30; (ii) the 3-7 lock switch (E3.28 and K7.43): Distance between Nξ of K7.43 and Cδ of E3.28; (iii) The transmission switch or the rotamer toggle switch (W6.48 and F6.44): The center of mass distance between the side chains of L3.40 and W6.48 as a measure of the conformational change from the inactive to active state; (iv) The tyrosine microswitches (Y7.53 and Y5.58): Distance between the oxygen atoms of the hydroxyl groups of Y7.53

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and Y5.58. From these microswitches we see that our structures are generally closer to the active- than to the inactive-crystal structure, thus representing active-like states. The exception is in microswitch iv, that is Y7.53 and Y5.58, in which the obtained mean values are closer to the inactive-crystal structure in both 83-113-247 and 83-113-134-247-249 protonated states, thus showing that this microswitch is not a good indicator of activation in our simulations.

→ Figure 6

In addition to the above molecular microswitches, we have found (unpublished results) that a segment of residues on TM3, the so-called TM3 core, is particularly relevant in the transmission of the activation message along the TM domain. The TM3 core consists of L1253.40 to L1283.43 residues. A structural analysis on a set of Class A GPCR crystal structures showed (unpublished results) that this segment of residues presents interactions with all helices except TM1 and TM7 in both inactive and active/intermediate

states.

Interestingly,

these

interactions

are

reinforced

in

active/intermediate states by the formation of new interactions with TM7, TM6 and TM5 along an extended TM3 core which includes residues T1183.33 to L1283.43. These new interactions include L3.40-W6.48 and L3.43-N7.49, whose residues have been shown to be relevant for receptor activation in Class A GPCR.42,43,44 Activation of the TM3 core by the formation of these new interactions facilitates the connection between the extracellular and intracellular sides of the receptor and the delivery of the activation message from the ligand-recognition site to the G protein-binding pocket. To evaluate from our simulations whether the TM3 core conformation may be an indicator of receptor active/inactive-state, PMFs were calculated for various receptor protonated

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states using the RMSD between the TM3 core of the structures from the MD simulations and the active 3PQR crystal structure as a reaction coordinate. Figure S2 (Supplementary Information) shows the PMF curve for the combination of protonated residues that in our MD simulations generated structures closest to the active 3PQR crystal structure, i.e. the protonated 83-113-247 state. In addition and for comparison, the PMF curves for all the combinations of pairs of protonated states from the parent triad were plotted. The relative location of the minima is in agreement with our previous results indicating that the protonated 83-113-247 state is more prone to achieving an active state than the other combinations of protonated residues, thus indicating the relevance of the TM3 core in the process of receptor activation. Having identified the acidic residues whose protonation facilitates the activation of opsin, we aimed to investigate their individual contributions, while bearing in mind that receptor functioning cannot be considered as a simple algebraic sum of independent terms. Our MD simulations show that protonation of E1133.28 allows the separation between the extracellular ends of TM3 and TM7 (Figure S3 Supplementary Information), thus breaking the 3-7 lock microswitch. Protonation of E1133.28 has been proposed as the first activation switch in rhodopsin.38 In the inactive state, 11-cis retinal is covalently linked to K2967.43 via a PSB, with E1133.28 acting as a counterion. After light absorption by retinal, and its subsequent conformational isomerization to all-trans, there is a proton transfer from PSB to E1133.28,40 facilitating the breaking of the E1133.28-K2967.43 ionic interaction. With respect to D832.50, our MD simulations suggest that protonation of this residue favors the active conformation of the TM3 core, as indicated by the comparison of the RMSD between the TM3 core of the active crystal structure and the structures generated along a number of MD trajectories with different protonation states (Figure S2 Supplementary Information). It can be seen in Figure S2

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that the state not including protonation of D832.50 (the protonated 113-247 state) presents the highest values for the minima of RMSD curves, thus showing the effect that protonation of this residue has on receptor activation. A significant population of protonated D832.50 is guaranteed by its relatively high pKa value as shown by theoretical calculations (pKa’s of 9.37 and 8.76 were obtained for D83 in inactive 1GZM and active 3PQR rhodopsin crystal structures, respectively, by using the PROPKA program)39 and experimental spectroscopic data.38 As discussed above, protonation of D832.50 and E1133.28 is necessary to activate the receptor; however, in our study, activation of opsin was possible only when D832.50, E1133.28 and E2476.30 were simultaneously protonated. Protonation of E2476.30 leads to the breaking of the ionic lock, which is a necessary but not sufficient condition for receptor activation17 as it is possible to find some inactive Class A GPCR structures with a broken ionic lock (see for instance A2AR PDB code: 3EML45 and β2AR PDB code: 2RH1).46 Thus, we have found that the minimal set of protonated acidic residues bearing a combined driving force for opsin activation consists of E1133.28, D832.50 and E2476.30. These residues, situated at strategic positions along the TM domain and involving the 3-7 lock, the ionic lock and H3 core microswitches, seem to govern the process of opsin activation in an additive manner (Figure 6). An acidic residue in position 6.30 is conserved in only ∼30% of GPCRs, though hydrogen-bonding interactions between helices 3 and 6 as an alternative to the ionic lock have been proposed-47 and 3-7 lock interactions involving an acidic residue in position 3.28 seem to be a particular feature of rhodopsin. However, the structural and functional resemblances between GPCRs suggest that the hypotheses generated in this study can be extended, at least in part, to other Class A GPCRs. In this context, analysis of the structural and functional effects of protonation of particular acidic residues may provide

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new insights into the activation mechanism of GPCRs (see Ref.

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48

for a discussion on

the relationship between receptor active state and the protonic equilibria among receptor ionic species).

→ Figure 7

CONCLUSION In the present study some structural features characteristic of GPCR activation were observed for opsin through unbiased MD simulations performed on a regular workstation. In contrast to some other simulations in the literature, which used predefined reaction coordinates to achieve receptor activation (see Ref.

49

discussion) or specialised computer hardware for lengthy timescales (see Refs.

for

14,17

),

opsin activation was obtained in our simulations by protonation of key acidic residues and is achievable on readily-accessible timescales with conventional computational hardware. Protonation of residues D832.50, E1133.28, and E2476.30 was the main source of stabilization for the activated receptor although other residues, as for instance E1343.49, seem to also contribute to receptor activation.38,41 Neutralization of E1133.28 causes the separation between the extracellular ends of TM3 and TM7 by breaking the electrostatic interaction between E1133.28 and K2967.43. Protonation of D832.50 is of relevance to promoting the active conformation of the TM3 core, a segment of residues including L1253.40 to L1283.43. Finally, neutralization of E2476.30 causes the breaking of the R135E247 ionic lock facilitating the outward movement of TM6. The combined protonation of D832.50, E1133.28 and E2476.30 seems to constitute a set of coordinated molecular microswitches bearing a joint driving force for receptor activation. Analysis of receptor activation by protonation of selected acidic residues may be applied to GPCRs, in

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general, to provide more insights on receptor activation mechanisms. MD simulations on receptor systems in which protonation states are varied systematically may yield a mechanistic map of the common and differential routes that GPCRs follow to achieve activation. These mechanistic proposals can be further validated by site-directed mutagenesis experiments. This plan seems reasonable as it has been recently proposed that GPCRs share membrane-spanning ionizable networks that seem characteristic of receptor activation.50

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Table 1. Comparison between structural properties (microswitches) associated to receptor activation between PMF minima closest to the active state in two selected opsin protonation states and inactive (1GZM) and active (3PQR) rhodopsin crystal structures 83-113-247 83-113-134-247-249 1GZM 3PQR n=98 n=75 1.390±0.001 1.070±0.001 0.48 1.79 Ionic lock R3.50-E6.30 0.599±0.004 0.809±0.005 0.38 0.64 3-7 lock 3.28 switch E K7.43 0.522±0.005 0.493±0.004 0.81 0.43 L3.40-W6.48 7.53 5.58 1.735±0.017 1.857±0.012 2.38 0.50 Y -Y Calculated sample values (n= 98 in the 83-113-247 state and n=75 in the 83-113-134247-249 state) are expressed as mean±SEM values. Ionic lock R3.50-E6.30 : Distance between Cξ of R3.50 and Cδ of E6.30. 3-7 lock switch E3.28-K7.43: Distance between Nξ of K7.43 and Cδ of E3.28. L3.40-W6.48: The transmission switch or the rotamer toggle switch (W6.48 and F6.44). In this case we chose the center of mass distance between the side chains of L3.40 and W6.48. Y7.53-Y5.58: The tyrosine microswitches (Y7.53 and Y5.58). Distance between the oxygen atoms of the hydroxyl groups of Y7.53 and Y5.58. All values are given in nm.

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FIGURE LEGENDS Figure 1. Scatter plot of d1 (the distance between Cξ of R1353.50 and Cδ of E2476.30) versus d2 (the distance between Cδ of E2496.32 and Cα of N3108.47) from the MD simulation of opsin with D832.50 and E1133.28 protonated (the protonated 83-113 state). MD production: 156 ns. Correlation coefficient R = -0.143. The green and red squares correspond to the active (3PQR) and inactive (1GZM) rhodopsin structures, respectively. Axes have been scaled to fixed values in Figures 1 to 4 for proper comparisons between plots.

Figure 2. Scatter plot of d1 (the distance between Cξ of R1353.50 and Cδ of E2476.30) versus d2 (the distance between Cδ of E2496.32 and Cα of N3108.47) from the MD simulation of opsin with A. D832.50, E1133.28 and E1343.49 protonated (the protonated 83-113-134 state). MD production: 80 ns. Correlation coefficient R = 0.109; B. D832.50, E1133.28 and E2496.32 protonated (the protonated 83-113-249 state). MD production: 74 ns. Correlation coefficient R = 0.107; C. D832.50, E1133.28 and E2476.30 protonated (the protonated 83-113-247 state). MD production: 54 ns. Correlation coefficient R = 0.833. The green and red squares correspond to the active (3PQR) and inactive (1GZM) rhodopsin structures, respectively. D. Structural snapshot of the MD simulation corresponding to plot C (green). The structure corresponds to a minimum with d1=1.39 nm and d2= 0.88 nm distance values. The crystallographic structure of active rhodopsin 3PQR (cyan) has been added for comparison. The computed opsin can accommodate the G protein moiety (yellow) present in the crystal structure.

Figure 3. Scatter plot of d1 (the distance between Cξ of R1353.50 and Cδ of E2476.30) versus d2 (the distance between Cδ of E2496.32 and Cα of N3108.47) from the MD

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simulation of opsin with A. D832.50, E1133.28, E2476.30 and E2496.32 protonated (the protonated 83-113-247-249 state). MD production: 57 ns. Correlation coefficient R = 0.081; B. D832.50, E1133.28, E1343.49 and E2476.30 protonated (the protonated 83-113134-247 state). MD production: 72 ns. Correlation coefficient R = -0.052. The green and red squares correspond to the active (3PQR) and inactive (1GZM) rhodopsin structures, respectively.

Figure 4. A. Scatter plot of d1 (the distance between Cξ of R1353.50 and Cδ of E2476.30) versus d2 (the distance between Cδ of E2496.32 and Cα of N3108.47) from the MD simulation of opsin with D832.50, E1133.28, E1343.49, E2476.30 and E2496.32 protonated (the protonated 83-113-134-247-249 state). MD production: 64 ns. Correlation coefficient R = 0.710. The green and red squares correspond to the active (3PQR) and inactive (1GZM) rhodopsin structures, respectively. B. Structural snapshot of the MD simulation corresponding to plot A (pink). The structure corresponds to a minimum with d1= 1.07 nm and d2= 0.92 nm distance values. The crystallographic structure of active rhodopsin 3PQR (cyan) has been added for comparison. The computed opsin can accommodate the G protein moiety (yellow) present in the crystal structure.

Figure 5. Scatter plot of d1 (the distance between Cξ of R1353.50 and Cδ of E2476.30) versus d2 (the distance between Cδ of E2496.32 and Cα of N3108.47) from the MD simulation of opsin with A. D832.50, E1133.28 and E2476.30 protonated (the protonated 83-113-247 state). MD production: 260 ns. Correlation coefficient R = 0.65; B. the unprotonated acidic residue state. MD production: 260 ns. Correlation coefficient R = 0.39. In both A and B plots the green and red squares correspond to the active (3PQR) and inactive (1GZM) rhodopsin structures, respectively. C. Structural snapshot of the

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MD simulation corresponding to plot A (green). The structure corresponds to a minimum with d1 = 1.35 nm and d2 = 0.88 nm distance values. The crystallographic structure of active rhodopsin 3PQR (cyan) has been added for comparison. The computed opsin can accommodate the G protein moiety (yellow) present in the crystal structure. D. Structural snapshot of the MD simulation corresponding to plot B (rose). The structure corresponds to a minimum with d1 = 1.07 nm and d2 = 0.44 nm distance values. The crystallographic structure of inactive rhodopsin 1GZM (violet) has been added for comparison.

Figure 6. A. B. PMF (bin size= 0.02 nm) of the MD simulation of opsin including three protonated residues: D832.50, E1133.28 and E2476.30 (production run: 54 ns), solid line and five protonated residues: D832.50, E1133.28, E1343.49, E2476.30 and E2496.32 (production run: 64 ns), dashed line. A. The distance d1 between Cξ of R1353.50 and Cδ of E2476.30 (the ionic lock switch) was used as the reaction coordinate. B. The distance d2 between Cδ of E2496.32 and Cα of N3108.47 was used as the reaction coordinate.

Figure 7. D832.50, E1133.28 and E2476.30 constitute the minimum ensemble of acidic residues whose protonation facilitates opsin activation.

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ACKNOWLEDGMENTS This study was supported in part by Ministerio de Economía y Competitividad (Grants SAF2010-19257 and ERA-NET NEURON PCIN-2013-018-C03-02) and Fundació La Marató de TV3 (Refs 110230). J.G. participates in the European COST Action CM1207 (GLISTEN: GPCR-Ligand Interactions, Structures, and Transmembrane Signalling: a European Research Network).

CONFLICT OF INTEREST There is no conflict of interest.

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References (1) Katritch, V.; Cherezov, V.; Stevens, R. C. Structure-Function of the G ProteinCoupled Receptor Superfamily. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 531556. (2) Palczewski, K.; Orban, T. From Atomic Structures to Neuronal Functions of G Protein-Coupled Receptors. Annu. Rev. Neurosci. 2013, 36, 139-164. (3) Overington, J. P.; Al Lazikani, B.; Hopkins, A. L. How Many Drug Targets Are There? Nat. Rev. Drug Discov. 2006, 5, 993-996. (4) Rask-Andersen, M.; Almen, M. S.; Schioth, H. B. Trends in the Exploitation of Novel Drug Targets. Nat. Rev. Drug Discov. 2011, 10, 579-590. (5) Lagerstrom, M. C.; Schioth, H. B. Structural Diversity of G Protein-Coupled Receptors and Significance for Drug Discovery. Nat. Rev. Drug Discov. 2008, 7, 339-357. (6) Kobilka, B. K. G Protein Coupled Receptor Structure and Activation. Biochim. Biophys. Acta 2007, 1768, 794-807. (7) Zhang Lab. GPCR-EXP for Experimentally Solved GPCR Structures. http://zhanglab.ccmb.med.umich.edu/GPCR-EXP/ (accessed July 3, 2015). (8) Venkatakrishnan, A. J.; Deupi, X.; Lebon, G.; Tate, C. G.; Schertler, G. F.; Babu, M. M. Molecular Signatures of G-Protein-Coupled Receptors. Nature 2013, 494, 185-194. (9) Wang, C.; Wu, H.; Katritch, V.; Han, G. W.; Huang, X. P.; Liu, W.; Siu, F. Y.; Roth, B. L.; Cherezov, V.; Stevens, R. C. Structure of the Human Smoothened Receptor Bound to an Antitumour Agent. Nature 2013, 497, 338-343. (10) Hollenstein, K.; Kean, J.; Bortolato, A.; Cheng, R. K.; Dore, A. S.; Jazayeri, A.; Cooke, R. M.; Weir, M.; Marshall, F. H. Structure of Class B GPCR Corticotropin-Releasing Factor Receptor 1. Nature 2013, 499, 438-443. (11) Siu, F. Y.; He, M.; de, G. C.; Han, G. W.; Yang, D.; Zhang, Z.; Zhou, C.; Xu, Q.; Wacker, D.; Joseph, J. S., et al. Structure of the Human Glucagon Class B GProtein-Coupled Receptor. Nature 2013, 499, 444-449. (12) Wu, H.; Wang, C.; Gregory, K. J.; Han, G. W.; Cho, H. P.; Xia, Y.; Niswender, C. M.; Katritch, V.; Meiler, J.; Cherezov, V., et al. Structure of a Class C GPCR Metabotropic Glutamate Receptor 1 Bound to an Allosteric Modulator. Science 2014, 344, 58-64. (13) Dore, A. S.; Okrasa, K.; Patel, J. C.; Serrano-Vega, M.; Bennett, K.; Cooke, R. M.; Errey, J. C.; Jazayeri, A.; Khan, S.; Tehan, B., et al. Structure of Class C GPCR Metabotropic Glutamate Receptor 5 Transmembrane Domain. Nature 2014, 511, 557-562.

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(14) Nygaard, R.; Zou, Y.; Dror, R. O.; Mildorf, T. J.; Arlow, D. H.; Manglik, A.; Pan, A. C.; Liu, C. W.; Fung, J. J.; Bokoch, M. P., et al. The Dynamic Process of Beta(2)-Adrenergic Receptor Activation. Cell 2013, 152, 532-542. (15) Huber, T.; Sakmar, T. P. Chemical Biology Methods for Investigating G Protein-Coupled Receptor Signaling. Chem Biol 2014, 21, 1224-1237. (16) Lohse, M. J.; Maiellaro, I.; Calebiro, D. Kinetics and Mechanism of G ProteinCoupled Receptor Activation. Curr. Opin. Cell Biol 2013, 27C, 87-93. (17) Dror, R. O.; Arlow, D. H.; Maragakis, P.; Mildorf, T. J.; Pan, A. C.; Xu, H.; Borhani, D. W.; Shaw, D. E. Activation Mechanism of the Beta2-Adrenergic Receptor. Proc. Natl. Acad. Sci. U. S. A 2011, 108, 18684-18689. (18) Lee, S.; Bhattacharya, S.; Grisshammer, R.; Tate, C.; Vaidehi, N. Dynamic Behavior of the Active and Inactive States of the Adenosine A(2A) Receptor. J Phys. Chem B 2014, 118, 3355-3365. (19) Dalton, J. A.; Lans, I.; Giraldo, J. Quantifying Conformational Changes in GPCRs: Glimpse of a Common Functional Mechanism. BMC. Bioinformatics. 2015, 16, 124. (20) Nygaard, R.; Frimurer, T. M.; Holst, B.; Rosenkilde, M. M.; Schwartz, T. W. Ligand Binding and Micro-Switches in 7TM Receptor Structures. Trends Pharmacol Sci. 2009, 30, 249-259. (21) Deupi, X.; Standfuss, J. Structural Insights into Agonist-Induced Activation of G-Protein-Coupled Receptors. Curr. Opin. Struct. Biol. 2011, 21, 541-551. (22) Trzaskowski, B.; Latek, D.; Yuan, S.; Ghoshdastider, U.; Debinski, A.; Filipek, S. Action of Molecular Switches in GPCRs--Theoretical and Experimental Studies. Curr. Med. Chem. 2012, 19, 1090-1109. (23) Ballesteros, J. A.; Weinstein, H. Integrated Methods for the Construction of Three-Dimensional Models and Computational Probing of Structure-Function Relations in G Protein-Coupled Receptors. In Methods in Neurosciences; Academic Press, Inc.: 1995; Vol. 25, Chapter 19. (24) Choe, H. W.; Park, J. H.; Kim, Y. J.; Ernst, O. P. Transmembrane Signaling by GPCRs: Insight From Rhodopsin and Opsin Structures. Neuropharmacology 2011, 60, 52-57. (25) Deupi, X. Relevance of Rhodopsin Studies for GPCR Activation. Biochim. Biophys. Acta 2014, 1837, 674-682. (26) Han, M.; Smith, S. O.; Sakmar, T. P. Constitutive Activation of Opsin by Mutation of Methionine 257 on Transmembrane Helix 6. Biochemistry 1998, 37, 8253-8261. (27) Park, J. H.; Scheerer, P.; Hofmann, K. P.; Choe, H. W.; Ernst, O. P. Crystal Structure of the Ligand-Free G-Protein-Coupled Receptor Opsin. Nature 2008, 454, 183-187.

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(28) Scheer, A.; Fanelli, F.; Costa, T.; de Benedetti, P. G.; Cotecchia, S. The Activation Process of the Alpha1B-Adrenergic Receptor: Potential Role of Protonation and Hydrophobicity of a Highly Conserved Aspartate. Proc. Natl. Acad. Sci. U. S. A 1997, 94, 808-813. (29) Seibert, C.; Harteneck, C.; Ernst, O. P.; Schultz, G.; Hofmann, K. P. Activation of the Rod G-Protein Gt by the Thrombin Receptor (PAR1) Expressed in Sf9 Cells. Eur. J Biochem. 1999, 266, 911-916. (30) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. J. Chem. Theor. Comput. 2008, 4, 435-447. (31) Oostenbrink, C.; Villa, A.; Mark, A. E.; van Gunsteren, W. F. A Biomolecular Force Field Based on the Free Enthalpy of Hydration and Solvation: the GROMOS Force-Field Parameter Sets 53A5 and 53A6. J. Comput Chem. 2004, 25, 1656-1676. (32) Li, J.; Edwards, P. C.; Burghammer, M.; Villa, C.; Schertler, G. F. Structure of Bovine Rhodopsin in a Trigonal Crystal Form. J. Mol. Biol 2004, 343, 14091438. (33) Berger, O.; Edholm, O.; Jahnig, F. Molecular Dynamics Simulations of a Fluid Bilayer of Dipalmitoylphosphatidylcholine at Full Hydration, Constant Pressure, and Constant Temperature. Biophys. J. 1997, 72, 2002-2013. (34) Kandt, C.; Ash, W. L.; Tieleman, D. P. Setting Up and Running Molecular Dynamics Simulations of Membrane Proteins. Methods 2007, 41, 475-488. (35) Choe, H. W.; Kim, Y. J.; Park, J. H.; Morizumi, T.; Pai, E. F.; Krauss, N.; Hofmann, K. P.; Scheerer, P.; Ernst, O. P. Crystal Structure of Metarhodopsin II. Nature 2011, 471, 651-655. (36) Vanni, S.; Neri, M.; Tavernelli, I.; Rothlisberger, U. A Conserved ProtonationInduced Switch Can Trigger "Ionic-Lock" Formation in Adrenergic Receptors. J. Mol. Biol. 2010, 397, 1339-1349. (37) Ghanouni, P.; Schambye, H.; Seifert, R.; Lee, T. W.; Rasmussen, S. G.; Gether, U.; Kobilka, B. K. The Effect of PH on Beta(2) Adrenoceptor Function. Evidence for Protonation-Dependent Activation. J. Biol. Chem. 2000, 275, 3121-3127. (38) Mahalingam, M.; Martinez-Mayorga, K.; Brown, M. F.; Vogel, R. Two Protonation Switches Control Rhodopsin Activation in Membranes. Proc. Natl. Acad. Sci U. S. A 2008, 105, 17795-17800. (39) Olsson, M. H. M.; Søndergaard, C. R.; Rostkowski, M.; Jensen, J. H. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical PKa Predictions. J. Chem. Theory Comput. 2011, 7, 525-537.

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(40) Jager, F.; Fahmy, K.; Sakmar, T. P.; Siebert, F. Identification of Glutamic Acid 113 As the Schiff Base Proton Acceptor in the Metarhodopsin II Photointermediate of Rhodopsin. Biochemistry 1994, 33, 10878-10882. (41) Vogel, R.; Mahalingam, M.; Ludeke, S.; Huber, T.; Siebert, F.; Sakmar, T. P. Functional Role of the "Ionic Lock"--an Interhelical Hydrogen-Bond Network in Family A Heptahelical Receptors. J Mol. Biol. 2008, 380, 648-655. (42) Holst, B.; Nygaard, R.; Valentin-Hansen, L.; Bach, A.; Engelstoft, M. S.; Petersen, P. S.; Frimurer, T. M.; Schwartz, T. W. A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-Transmembrane Receptors. J Biol. Chem 2010, 285, 3973-3985. (43) Valentin-Hansen, L.; Holst, B.; Frimurer, T. M.; Schwartz, T. W. PheVI:09 (Phe6.44) As a Sliding Microswitch in Seven-Transmembrane (7TM) G ProteinCoupled Receptor Activation. J. Biol Chem. 2012, 287, 43516-43526. (44) Bhattacharya, S.; Vaidehi, N. Differences in Allosteric Communication Pipelines in the Inactive and Active States of a GPCR. Biophys. J. 2014, 107, 422-434. (45) Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y.; Lane, J. R.; Ijzerman, A. P.; Stevens, R. C. The 2.6 Angstrom Crystal Structure of a Human A2A Adenosine Receptor Bound to an Antagonist. Science 2008, 322, 1211-1217. (46) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K., et al. HighResolution Crystal Structure of an Engineered Human Beta2-Adrenergic G Protein-Coupled Receptor. Science 2007, 318, 1258-1265. (47) Bhattacharya, S.; Hall, S. E.; Li, H.; Vaidehi, N. Ligand-Stabilized Conformational States of Human Beta(2) Adrenergic Receptor: Insight into GProtein-Coupled Receptor Activation. Biophys. J 2008, 94, 2027-2042. (48) Giraldo, J. A PH-Dependent Model of the Activation Mechanism of the Histamine H2 Receptor. Biochem. Pharmacol. 1999, 58, 343-353. (49) Miao, Y.; Nichols, S. E.; McCammon, J. A. Free Energy Landscape of GProtein Coupled Receptors, Explored by Accelerated Molecular Dynamics. Phys. Chem Chem Phys. 2014, 16, 6398-6406. (50) Isom, D. G.; Dohlman, H. G. Buried Ionizable Networks Are an Ancient Hallmark of G Protein-Coupled Receptor Activation. Proc. Natl. Acad. Sci U. S. A 2015.

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Toc graphics

Active-like Opsin

Extracellular domain 1.6

1.4

E113

TM6 TM3

D83 TM2

E247

Glu249(Cδ )-Asn310(Cα )(nm)

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The Journal of Physical Chemistry

1.2

1.0

0.8

0.6

0.4

0.2

Intracellular domain

Protonated Opsin Transversal point of view

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Arg135(Cξ)-Glu247(Cδ)(nm)

Inactive Opsin

31 Environment ACS Paragon Plus

1.8

2.0

The Journal of Physical Chemistry

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Figure 1 129x90mm (150 x 150 DPI)

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The Journal of Physical Chemistry

Figure 2 214x169mm (150 x 150 DPI)

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The Journal of Physical Chemistry

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Figure 3 219x83mm (150 x 150 DPI)

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The Journal of Physical Chemistry

Figure 4 220x86mm (150 x 150 DPI)

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The Journal of Physical Chemistry

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Figure 5 197x156mm (150 x 150 DPI)

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The Journal of Physical Chemistry

Figure 6 226x83mm (150 x 150 DPI)

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The Journal of Physical Chemistry

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Figure 7 136x170mm (150 x 150 DPI)

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