Fig 1.
Reweighted Na+ density heat maps of simulated active and inactive MOR systems.
Residues D1473.32, D1142.50 and Y3367.53 are displayed as sticks to roughly indicate the locations of the orthosteric ligand binding site, the Na+ allosteric binding site, and the Na+ density gap (in the simulated inactive MOR system only), respectively, in the (a) inactive MOR crystal structure, (b) active MOR crystal structure with charged D2.50, and (c) active MOR crystal structure with protonated D2.50. Dotted lines symbolize the location of lipid head groups. A blue-to-red color scale illustrates low-to-high Na+ densities.
Fig 2.
Integrated free energy profiles of Na+ translocation through inactive MOR (green) and active MOR with either charged or protonated D1142.50 (red and brown, respectively), as well as at pH = 7 (orange), as a function of the Na+ z-coordinate.
The errors of the free energy are obtained via a bootstrapping procedure as described in the Methods.
Fig 3.
Transition networks formed by the metastable states obtained from the Markov state models constructed for the inactive MOR (panel a) and active MOR with charged or protonated D1142.50 (panels b and c, respectively).
States are labeled according to the Na+ position relative to the receptor as extracellular or cytoplasmic states, while bound states are highlighted with a black contour. The size of the circles depicting the metastable states is proportional to the probability of individual states and the thickness of the arrows connecting them is proportional to the transition flux between the states. Three-dimensional structures are shown in insets for each metastable state and represent the center of the corresponding PCCA cluster. Residues highlighted as sticks are the highly coupled residues from tICA with the most significant sidechain conformational change among different PCCA cluster center structures based on visual inspection.
Fig 4.
Estimated transition times of (a) Na+ binding (from extracellular to bound states, in ns), (b) Na+ dissociation (from bound to extracellular states, in μs), and (c) Na+ egress (from bound to intracellular states, in s) as a function of extracellular Na+ concentration.
The Na+ concentration in the cytoplasm is assumed to be constant. Transition times are calculated as the median of the mean first passage times calculated from individual bootstrap samples and the full sample. Confidence intervals were estimated as the differences from the 1st and 3rd quartiles.
Fig 5.
Percent change in [3H]-DAMGO binding as a function of increasing concentrations of NaCl, reported for a concentration of [3H]-DAMGO of 1 nM, and assuming a [3H]-DAMGO binding constant of 4 nM.
Circles represent values from radioligand binding experiments, the solid line represents the predictions from the simulation data while the shaded region corresponds to the errors associated with the predictions. Binding values are the means ± s.e.m. of three independent experiments. Error bars that are not visible are smaller than the size of the symbol.