Dark Rhod Meta I Meta-II 12 13 14 16 26 27 28 30 31 32 44 98 101 103 125 176 178 179 181 182 183 185 186 187 188 189 190 191 192 193 194 212 276 284 285 Retinal Contacts • 7 transmembrane (7TM) α-helical proteins • 800 GPCRs in the human genome • Binding of G protein to the active form of the receptor induces: - Conformational change - Signal transduction across membrane - Signaling cascade • Biomedically relevant - More than 30% drugs target GPCRs • Rhodopsin is the mammalian dim- light receptor - Retinal: covalently bound - 11-cis: inverse agonist - All-trans: agonist •Box size: 74 Å x 74 Å x 90Å •Forcefield: CHARMM27/36 - Retinal parameters obtained from the Feller lab •Ensemble: NPγT - γ = 30 dyn/cm • Helix 6 principal axis cosine with membrane normal - Cytoplasmic half of the helix - Opsin and Meta II more tilted, broader range • Other ligands - Better agonists - Weaker inverse agonists • Extend accessible timescales with simple models - Structure-based potentials - Transitions in equilibrium Given their function as tranducers of molecular signals across the cell membrane, G protein-coupled receptors (GPCRs) constitute a major target for drugs in a wide variety of physiological scenarios. Understanding the course of structural transitions that allosterically modulate their activation is therefore fundamental towards improving rational drug design. Here, we analyze unbiased microsecond-scale all-atom molecular dynamics simulations to characterize distinct ensembles of the class A GPCR rhodopsin that correspond to both active- and inactive-like conformations, in the presence and absence of the ligand. By monitoring the ligand's orientation and interactions within the binding pocket, we show that retinal adopts heterogeneous conformations that are consistent with ensemble- dependent dynamics. • PCA calculated on aggregate basis set - All 5 ensembles - Transmembrane Cα's only - Trajectories downsampled to 5 ns resolution • Average displacement by ensemble - PC1 distinguishes active-like from inactive - Amplitude of fluctuations: diversity • System size: ~46,000 atoms - 123 SDPE lipids - ~ 8,000 waters - 100 mM NaCl • Internal solvation - H-bonding patterns by ensemble ‧ Water and salt • Lipid-protein interactions • Predict 2 H NMR spectra for retinal • Enhanced sampling using Markov State Models • Thermostat: Langevin - 310 K - 1 bar •Electrostatics: PME • VDW cutoff: 10 Å •Timestep: 2 fs - RATTLE • Software: - NAMD 2.8 - BlueGene/Q • Ensemble projected onto PC1 and PC2 - Every dot represents a single structure in the corresponding ensemble • Opsin overlaps with all four en- sembles • Dark Rhod and Meta II are compact • Meta I explores some unique states • One representative time series per ensemble - Residue number colored by helix - Ensemble-dependent interactions - Contacts vary on the timescale of hundreds of ns • Average occupancy - Residue number colored by helix - Meta I is the most heterogeneous • Retinal changes during acti- vation - More transient contacts in Meta I - Prefered orientation switches upon isomerization - Retinal elongates in Meta I and Meta II - Retinal dynamics are strongly coupled to protein motions • Transitions involve protein's most collective motions • H6 orientation and contacts are ensemble-dependent • Overall slow relaxation times - Events require hundreds of ns - Multiple simulations essential • Transient intermediates • Meta I and Meta II are in equilibrium - Only Meta II activates G protein • Opsin is apo form - Crystal looks active-like - Experimentally has minimal activity • Dark Opsin is a proposed inactive-like, ligand-free state • Retinal isomerizes from 11-cis to all-trans when a photon is captured by dark-state rhodopsin. • To understand state-dependent dynamics and their correlation with retinal motions we analyzed the following all-atom MD simulations: • Cosine of methyl orientation with membrane normal - Meta II and Dark Rhod: retinal orien- tations distinctly polarized near C5 and C13 - Meta I: more heterogeneous confor- mations • Correlation between rhodopsin and retinal's most collective motions - Principal component analysis (PCA) - Each trajectory of every ensemble for ligand and receptor - Alignment on transmembrane Cα's • Representative time series of PC1 for protein and retinal • Protein-retinal correlation per ensemble - Compute PCA for protein and retinal for each trajectory - Correlation between PC1 for each - Average over 6 trajectories per ensemble - Differences are not statistically significant (p-value > 0.05) - Retinal remains strongly coupled • Retinal-protein interactions - Contact: Residue-retinal centroid- to-centroid distance < 8 Å - Retinal taken as a single residue • Ilustration: Five highest occupancy contacts - Ballesteros-Weinstein numbering in superscript • C3-NZ distance vs. time - Ensemble average over 6 trajec- tories Meta I: all-trans • Average helix 6 interactions - Contact: Centroid-to-centroid distance < 10 Å - Helix 6 taken residue by residue Abstract Class A GPCR Rhodopsin Retinal Dynamics Distinguish Protein State Simulation Details Large-Scale Protein Motions Vary Between Ensembles Future Directions Retinal-Protein Contacts Conclusions Retinal Orientation Varies with Ensemble Retinal Motions Correlate with Overall Protein Dynamics Helix 6 Dynamics Characterize Rhodopsin Ensembles Ensemble-Average Contacts Dynamic Ligand-Protein Interactions Alter Rhodopsin's Conformational Ensemble: Simulations of Rhodopsin and Opsin Dynamic Ligand-Protein Interactions Alter Rhodopsin's Conformational Ensemble: Simulations of Rhodopsin and Opsin Letty Salas-Estrada, Nicholas Leioatts, Tod D. Romo, Shairy Azmy Danial, Alan Grossfield University of Rochester Medical School, Rochester, NY, USA Letty Salas-Estrada, Nicholas Leioatts, Tod D. Romo, Shairy Azmy Danial, Alan Grossfield University of Rochester Medical School, Rochester, NY, USA Rhodopsin Photocycle Intracellular Extracellular (1) Grossfield et al., J. Mol. Biol. 2008, 381: 478-486 T118 3.33 G121 3.36 M207 5.42 H211 5.46 F212 5.47 W265 6.48 Y268 6.51 A269 6.52 Retinal Dark Rhod Meta I Meta II * : p-value vs. Meta II < 0.05 0 0.2 0.4 0.6 0.8 1 |Correlation| Dark Rhod Meta I Meta−II Meta II Meta I Dark Rhod Opsin Dark Opsin Dark Rhod Dark Opsin? Opsin Meta II Meta I Bathorhodopsin Lumirhodopsin all-trans retinal retinal cis-trans isomerization 11-cis retinal photon Ensemble Structure Simulation Time (µs) Dark Rhod 1U19 6 runs x 1.8 Meta I previous work (1) 6 runs x 6.0 Meta II 3PXO 6 runs x 4.5 Opsin 3CAP 6 runs x 4.5 Dark Opsin 1U19 (ligand removed) 6 runs x 4.5 Total ≈ 130 µs C5 C9 C13 C5 C9 C13 Data analysis was performed using LOOS (Lightweight Object-Oriented Structure library), an open source C++ library designed by the Grossfield lab. LOOS is adaptable and compatible with all major simulation packages, providing a leveled and friendly platform for developing analysis applications. The source code is available at: http://loos.sourceforge.net Retinal Elongation 12 12.5 13 13.5 14 14.5 15 15.5 0 1000 2000 3000 4000 5000 6000 Elongation (Å) Time (ns) Meta I Meta II Dark Rhod • Retinal rapidly elongates after isomerization Retinal 0 0.1 0.2 0 0.1 0.2 Population 0 0.1 0.2 −1.0 −0.5 0.0 0.5 1.0 cos Θ Meta I Meta II Dark Rhod 0 1000 2000 Time (ns) 12 13 14 16 26 27 28 30 31 101 176 178 179 181 182 183 185 186 187 188 189 190 191 192 193 284 0 Retinal Contacts Dark Rhod 0 1000 2000 3000 4000 5000 6000 Time (ns) Meta I 0 1000 2000 3000 4000 Time (ns) Meta II *: p-value vs. Dark Rhod < 0.05 http://tinyurl.com/rtn-rhod-dyna Poster PDF −1 −0.5 0 0.5 1 −2 −1 0 1 2 0 500 1000 1500 Time (ns) Protein Retinal PC1 Displacement (Å) 0 1000 2000 3000 4000 5000 6000 Time (ns) 0 1000 2000 3000 4000 Time (ns) Dark Rhod Meta I Meta II −1.5 −1 −0.5 0 0.5 1 1.5 2 0 1000 2000 3000 4000 5000 6000 PC1 Displacement (Å) Time (ns) −2.5 −2 −1.5 −1 −0.5 0 0.5 1 1.5 −1.5 −1 −0.5 0 0.5 1 1.5 2 PC2 Displacement (Å) PC1 Displacement (Å) 0 0.2 0.4 0.6 0.8 1 Dark Rhod Meta I Meta-II Opsin Dark Opsin M1 N2 G3 T4 E5 G6 P7 N8 F9 Y10 V11 P12 F13 S14 N15 K16 K66 K67 L68 C140 K141 M143 S144 N145 W175 S176 R177 Y178 E181 G182 M183 G188 I189 D190 Y191 Y192 T193 P194 H195 E196 E197 A235 Q236 Q237 Q238 E239 S240 A241 T242 T243 Q244 K245 H278 Q279 G280 S281 D282 F283 G284 M309 N310 K311 Q312 F313 R314 N315 C316 M317 V318 T319 L321 C322 C323 K325 N326 H6 Contacts * * * * * * * * * * * * * * * * * * * Meta II Meta I Dark Rhod Opsin Dark Opsin 0 0.1 0.2 0.3 0.4 0.7 0.8 0.9 1 Population cos Θ Dark Rhod: 11-cis Meta II: all-trans Residues Contacting Retinal Residues Contacting Retinal Residues Contacting Helix 6 Dark Rhod Meta I Meta II Opsin Dark Opsin