Contact surfaces (shown for one protomer in dimeric arrangements) at the values of r indicated by dashed grey lines in Figure 2.

<p>The crystal structure contact surfaces are shown (on grey background) and additional distances are r = 3.12 nm, r = 3.64 nm and r = 4.60 nm for TM5,6/TM5,6 MOPr interface, and r = 3.80 nm, r = 4.00 nm and r = 4.60 nm for MOPr and KOPr at TM1,2,H8/TM1,2,H8, with the addition of r = 5.40 nm for KOPr.</p

PMFs calculated from the umbrella sampling, coarse-grained simulations for each of the different interfaces: MOPr TM1,2,H8/TM1,2,H8 is shown in red (error bars in pink), MOPr TM5,6/TM5,6 is shown in blue (error bars in light blue), and KOPr TM1,2,H8/TM1,2,H8 is in green (error bars in light green).

<p>The grayed region denoted ‘monomer’ is that in which the PMF curves were aligned to free-energy = 0. The dashed lines, shown at r = 3.12 nm, 3.64 nm, 3.80 nm, 4.00 nm, 4.60 nm, and 5.40 nm, indicate the values of separation at which the inter-protomer contacts were assessed.</p

Key results and methodological aspects of PMF curves in Figure 2.

<p>Key results and methodological aspects of PMF curves in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090694#pone-0090694-g002" target="_blank">Figure 2</a>.</p

Interacting residues at key values of separation (r) in the PMF (shown by dashed lines in Figure 2).

<p>Cut-off value is shortest inter-protomer contact distance less than 4.8 Å between any atoms, for representative structures from the different simulation windows indicated by the dashed lines. Bolded residues are those present in the crystallographic KOPr and MOPr structures at the same cut-off values. *The interaction with this residue comes from the palmitoyl chain of the C3.55, which is not present in the crystallographic structures. Italicized residues are mentioned specifically in the text.</p

The crystallographic structures of the opioid receptors indicate putative interfacial interactions.

<p>A) Side view of crystallographic dimers. MOPr protomers interacting at a TM1,2,H8/TM1,2,H8 interface colored in red and pink from PDB ID: 4DKL. MOPr protomers interacting at a TM5,6/TM5,6 interface in blue and light blue, also from PDB ID 4DKL, and KOPr protomers interacting at a TM1,2,H8/TM1,2,H8 interface are shown in green and light green, from PDB ID: 4DJH. T4L is shown in gray in all cases. B) Extracellular view of crystallographic dimers, colored as above. Cyan overlay shows collective variables used to define the interface between protomer 1 and protomer 2 (P1 and P2). The PMF is calculated as a function of the separation <i>(r)</i> between the COMs of the TM regions of the two protomers. The orientation of the interface was maintained during the simulations by two harmonic restraints centered on the values of the angles, α and β. The angle α is the projection on the x, y plane of the angle calculated between the COM of the helix defining the interface, the COM of the helical bundle of the protomer bearing this helix (P1) and the COM of the adjacent protomer (P2). Angle β is the equivalent angle for the adjacent protomer.</p

Hypothetical arrangements of B2AR dimers interacting with the Gs protein heterotrimer.

<p>The extracellular view of the B2AR-Gs protein complex (PDB ID: 3SN6) is shown with B2AR in grey cartoon representation, and the Gs heterotrimer, is shown in both cartoon and transparent surface (α is in orange, β is in cyan and γ is in pale blue). The first protomer of each of our minimum energy dimers for B2AR (Θ2 for TM4/3) is superimposed on the B2AR from the crystal structure 3SN6, and the position of the second protomer is shown in a green cartoon representation. An interface involving TM4 would favor an exclusive interaction of the dimer with the GαsAH subunit of the G protein, very close to the Gβ subunit when the interface is TM4/3 (panel A). In contrast, with TM1/H8 at the interface, the second protomer would not be involved in significant interactions with any of the G-protein subunits (in B).</p

Atomistic structural representations of representative minima of B1AR and B2AR homodimers for the different interfaces.

<p>Panel A shows a view from the cytoplasmic side of the minima Θ1 (dark shades) and Θ2 (lighter shades) for the B1AR (red/pink) and B2AR (blue/light blue) at the TM4/3 interface. The receptors are represented as helices with the loops removed for clarity, and are aligned on the left-most protomer. This panel highlights the small difference between the structures at the same separation but with slightly different angle minima. Panel B shows a view from the cytoplasmic side, of the minima extracted from the FES for the TM1/H8 interface. B1AR is shown in red and B2AR is shown in blue. The intracellular loops are omitted for clarity, and H8 and TM1 are highlighted to indicate the packing of the helices at the interface. Contacting residues are listed in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002649#pcbi.1002649.s006" target="_blank">Table S1</a> and depicted in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002649#pcbi.1002649.s004" target="_blank">Fig. S4</a>.</p

Free energy surface (FES) as a function of protomer separation.

<p>Panel (A) shows results obtained for the TM4/3 interface of B1AR (red) and B2AR (blue) homodimers. The monomeric state was defined as being in the <i>r</i> = 4.5–4.8 nm range, and was assigned a free energy of zero. Panel (B) shows the FES as a function of the distance between the centers of mass of the protomers at the TM1 interface formed by (red) B1AR and (blue) B2AR, with the monomeric state assigned as <i>r</i> = 5.5–5.9 nm. The error bars are given for each case, and are calculated as described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002649#pcbi.1002649.s009" target="_blank">Text S1</a>. In each panel, the inset shows a schematic representation of the CVs (<i>r</i>, <i>θ_a</i> and <i>θ_b</i>) in each of the interface arrangements.</p

Results of the free energy calculations of adrenergic receptor dimers.

<p>Listed are the depths of the minima in the FES, along with the calculated dimerization constant (K_D), the standard free energy change ΔG_X°, and the estimated lifetime of each simulated B1AR and B2AR dimer. Confidence intervals are given in parentheses.</p

Assessing the Relative Stability of Dimer Interfaces in G Protein-Coupled Receptors

<div><p>Considerable evidence has accumulated in recent years suggesting that G protein-coupled receptors (GPCRs) associate in the plasma membrane to form homo- and/or heteromers. Nevertheless, the stoichiometry, fraction and lifetime of such receptor complexes in living cells remain topics of intense debate. Motivated by experimental data suggesting differing stabilities for homomers of the cognate human β1- and β2-adrenergic receptors, we have carried out approximately 160 microseconds of biased molecular dynamics simulations to calculate the dimerization free energy of crystal structure-based models of these receptors, interacting at two interfaces that have often been implicated in GPCR association under physiological conditions. Specifically, results are presented for simulations of coarse-grained (MARTINI-based) and atomistic representations of each receptor, in homodimeric configurations with either transmembrane helices TM1/H8 or TM4/3 at the interface, in an explicit lipid bilayer. Our results support a definite contribution to the relative stability of GPCR dimers from both interface sequence and configuration. We conclude that β1- and β2-adrenergic receptor homodimers with TM1/H8 at the interface are more stable than those involving TM4/3, and that this might be reconciled with experimental studies by considering a model of oligomerization in which more stable TM1 homodimers diffuse through the membrane, transiently interacting with other protomers at interfaces involving other TM helices.</p> </div