Fraction of the observed hetero-dimeric interfaces during five independent MD simulations of the μ-OR/δ-OR and δ-OR/κ-OR systems.

<p>Best estimate of the fraction of each hetero-dimeric arrangement and its (2.5%,97.5%) confidence intervals (in parenthesis) are calculated as reported in the Methods section.</p><p>Fraction of the observed hetero-dimeric interfaces during five independent MD simulations of the μ-OR/δ-OR and δ-OR/κ-OR systems.</p

Superposition of the highly frequent homo-dimer configurations of δ-OR onto the closest available crystal structures of parallel receptors.

<p>Specifically, these are: TM1,2,H8/TM1,2,H8, TM1,2/TM5,6, and TM4,5/TM5,6, in panels A (RMSD of 3.57 Å from 4DJH), B (RMSD of 6.48 Å from 3OE8), and C (RMSD of 6.62 Å from 3OE8), respectively.</p

An example of OR setup at different simulation times.

<p>Location of the 16 simulated receptor molecules in one of the five runs executed for the κ-OR system (run #1), at different simulation times, (specifically: 0, 2, 6, and 10 μs)</p

Preferred Supramolecular Organization and Dimer Interfaces of Opioid Receptors from Simulated Self-Association

<div><p>Substantial evidence in support of the formation of opioid receptor (OR) di-/oligomers suggests previously unknown mechanisms used by these proteins to exert their biological functions. In an attempt to guide experimental assessment of the identity of the minimal signaling unit for ORs, we conducted extensive coarse-grained (CG) molecular dynamics (MD) simulations of different combinations of the three major OR subtypes, i.e., μ-OR, δ-OR, and κ-OR, in an explicit lipid bilayer. Specifically, we ran multiple, independent MD simulations of each homomeric μ-OR/μ-OR, δ-OR/δ-OR, and κ-OR/κ-OR complex, as well as two of the most studied heteromeric complexes, i.e., δ-OR/μ-OR and δ-OR/κ-OR, to derive the preferred supramolecular organization and dimer interfaces of ORs in a cell membrane model. These simulations yielded over 250 microseconds of accumulated data, which correspond to approximately 1 millisecond of effective simulated dynamics according to established scaling factors of the CG model we employed. Analysis of these data indicates similar preferred supramolecular organization and dimer interfaces of ORs across the different receptor subtypes, but also important differences in the kinetics of receptor association at specific dimer interfaces. We also investigated the kinetic properties of interfacial lipids, and explored their possible role in modulating the rate of receptor association and in promoting the formation of filiform aggregates, thus supporting a distinctive role of the membrane in OR oligomerization and, possibly, signaling.</p></div

Persistence times and persistence-to-exchange time ratios of cholesterol molecules derived from the OR homo-dimer simulations.

<p>Specifically, persistence times of cholesterol calculated for δ-OR/δ-OR, κ-OR/κ-OR, and μ-OR/μ-OR are reported, in ns, in panels A, B, and C, respectively. Persistence-to-exchange time ratios of cholesterol around isolated δ-OR, κ-OR, and μ-OR are shown in panels D, E, and F, respectively. Approximate locations of the centers of mass of the seven transmembrane helices (TM1-TM7) are indicated with red labels.</p

Fraction of the observed homo-dimeric interfaces during five independent MD simulations of the μ-OR/μ-OR, δ-OR/δ-OR, and κ-OR/κ-OR systems.

<p>Best estimate of the fraction of each homo-dimeric arrangement and its (2.5%,97.5%) confidence intervals (in parenthesis) are calculated as reported in the Methods section.</p><p>Fraction of the observed homo-dimeric interfaces during five independent MD simulations of the μ-OR/μ-OR, δ-OR/δ-OR, and κ-OR/κ-OR systems.</p

Persistence times of lipid molecules around μ-OR crystallographic interfaces using data from previously published simulations.

<p>Panels A and B show the persistence times of lipid molecules that are adjacent to the simulated TM1,2,H8/TM1,2,H8 and TM5,6/TM5,6 crystal dimers, respectively. Approximate locations of the centers of mass of the seven transmembrane helices (TM1-TM7) are indicated with solid and outlined red labels for each protomer, respectively. The lipid-exposed region of the protein surface adjacent to the interface is schematically highlighted with a red dashed line.</p

Association rates of OR hetero-dimers formed during five independent MD simulations of the δ-OR/μ-OR and δ-OR/κ-OR systems.

<p>Estimated k_on rates (in μm²/s) along with (2.5,97.5) confidence intervals.</p><p>Association rates of OR hetero-dimers formed during five independent MD simulations of the δ-OR/μ-OR and δ-OR/κ-OR systems.</p