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. 2021 Nov 4;29(11):1312-1325.e3.
doi: 10.1016/j.str.2021.06.015. Epub 2021 Jul 15.

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

Affiliations

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

Wanling Song et al. Structure. .

Abstract

GPCRs have been shown to form oligomers, which generate distinctive signaling outcomes. However, the structural nature of the oligomerization process remains uncertain. We have characterized oligomeric configurations of the adenosine A2a receptor (A2aR) by combining large-scale molecular dynamics simulations with Markov state models. These oligomeric structures may also serve as templates for studying oligomerization of other class A GPCRs. Our simulation data revealed that receptor activation results in enhanced oligomerization, more diverse oligomer populations, and a more connected oligomerization network. The active state conformation of the A2aR shifts protein-protein association interfaces to those involving intracellular loop ICL3 and transmembrane helix TM6. Binding of PIP2 to A2aR stabilizes protein-protein interactions via PIP2-mediated association interfaces. These results indicate that A2aR oligomerization is responsive to the local membrane lipid environment. This, in turn, suggests a modulatory effect on A2aR whereby a given oligomerization profile favors the dynamic formation of specific supramolecular signaling complexes.

Keywords: A2a receptor; GPCR; Markov state models; lipids; molecular dynamics; oligomerization.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests. M.S.P.S. is on the journal advisory board.

Figures

None
Graphical abstract
Figure 1
Figure 1
A2aR oligomerization sampled by MD simulations using complex membranes (A) System setup of A2aR oligomerization simulations. The selected number of receptor molecules (pale blue) were randomly inserted into a mixed lipid membrane of area 45 × 45 nm2. Views of the systems from the extracellular and intracellular surfaces and in the cross-section are shown, with the lipid species present color coded. Details of the different simulation systems setups are listed in Table 1. (B) The time evolution of minimum distance between pairs of receptors (from a simulations trajectory in the inactive state nine-copy simulation ensemble), illustrating that both association and dissociation events occur within the timescale simulated (selected events are highlighted by bold traces). (C) The number of association (defined as the smoothed minimum distance of a pair of proteins coming closer than 0.75 nm) and dissociation (defined as the smoothed distance of a pair of proteins separating to further than 0.75 nm) events sampled in each trajectory for each of the 9-copy and 16-copy system ensembles. (D) The associations and dissociations have led to a dynamic equilibrium as illustrated by the time course of oligomer formation for the inactive state nine-copy, active state nine-copy, and active state nine-copy NoPIP2 simulations. Data averages from all trajectories in the same simulation system were plotted to illustrate the ensemble trend. See Figure S2 for a full list.
Figure 2
Figure 2
Oligomer distribution The oligomer distributions are shown for the final 20 μs in the 9-copy simulation (A), for the final 10 μs in the 9-copy NoPIP2 simulation (B) and for the final 20 μs in 16-copy simulations (C), estimated by counting the number of oligomers in the systems. The error bars (black) denote standard deviations along the trajectory time course.
Figure 3
Figure 3
Characterization of oligomer quaternary structures (A) The oligomer quaternary structures from the same simulation set were clustered to identify the various oligomeric configurations. For the calculation of oligomer residence time, durations of each configuration were collected and sorted (blue dots in the left panel). A normalized survival function as a function of Δt (blue dots in the right panel) were modeled based on the sorted durations. A biexponential (red dashed line in the right panel) was used to fit to the survival function to obtain koff. To estimate the confidence of the calculated koff values, survival functions based on bootstrapped durations were modeled (gray lines in the right panel) from which standard deviations were calculated. (B) The following metrics were used to describe the oligomer configurations: for dimers, two binding angles (θ1, θ2), such that each of the angles describes the relative position of the dimer interface to the principal axis of that monomer that is parallel to H8 in a clockwise direction; for trimers, the bending angle ϕ defined by the center of mass of the three monomers; and for tetramers and pentamers, the projected lengths D1 and D2 on their first and second principal axes.
Figure 4
Figure 4
Population profiles of oligomeric configurations in the nine-copy ensemble Distributions of oligomeric configuration metrics defined in Figure 3B are shown in (A) for dimers, (B) for trimers, (C) for tetramers, and (D) for pentamers.
Figure 5
Figure 5
Oligomeric configurations with the longest residence times in the nine-copy ensembles The dimeric (A) and trimeric (B) structures with the longest residence times in each conformational state are shown, with the backbone beads of the receptor in cyan surface and the mini Gs in brown. PIP2 molecules at the association interfaces are shown in ball and sticks with different colors. Below each of structures are the association interfaces noted in bold (ICL, intracellular loop; TM, transmembrane helix; A//B, interface between surface A and surface B), along with the average values of the descriptive metrics of oligomer configurations (see text and Figure 3B for details), and the oligomer residence time, which is followed by the cluster ID label in brackets. Also see Figures S3 and S4 and Table S1.
Figure 6
Figure 6
The influence of PIP2 on A2aR oligomerization (A) PIP2 binding sites on the active state A2aR. The six binding sites calculated using PyLipID (https://doi.org/10.5281/zenodo.4999404) are displayed using different colors. Amino acid residues in each binding site are shown as spheres with radii proportional to their PIP2 residence times in the active state nine-copy simulations. (B) A representative PIP2 pose bound to the strongest PIP2 binding site from the active state simulations (corresponding to the red spheres in A) shown as a back-mapped atomistic structure. Binding site residues are shown as green sticks, and the PIP2 molecule as slate-blue/orange/red sticks. Electrostatic interactions between the PIP2 head group and protein residues are indicated by yellow dashed lines. (C) Comparison of (left panel) PIP2 residence times versus residue number in active state PIP2-containing simulations (9-copy) with (right panel) PS residence times in the active state PIP2-free simulations (9-copy NoPIP2). (D) Comparison of the average residence time as a function of oligomeric order between the three membrane environments (PIP2-containing, 9-copy; PIP2-free, 9-copy; and PIP2-containing, 16 copy) for the three states of the A2aR. Bar heights denote the average residence time of the oligomeric configurations from the same oligomeric order and the error bars show the 95% interval of 1,000 bootstrapped samples. (E) Active state dimer configurations with the three longest residence time from PIP2-free simulations. The backbone beads of the receptor are shown in cyan. The PS molecules at the association interfaces are shown in ball and sticks with different colors. Below each of the structures are the association interfaces in bold, the average values of the binding angles, and the oligomer residence time, which is followed by the cluster ID label in brackets. Also see Figure S7.
Figure 7
Figure 7
Markov state models of A2aR oligomerization The models calculated the kinetics of the oligomerization by monitoring the evolution of A2aR oligomers in the membrane. The oligomerization states are labeled as Aa-Bb-Cc in which A, B, and C denote the oligomeric orders present in the membrane, and a, b, and c denote the number of oligomers of the corresponding order. The thickness of the arrows is proportional to the corresponding reaction rate (only reaction rates >50 s−1 are shown) and the size of the circles to the equilibrium distributions. Reaction rates were calculated as the reciprocal of the corresponding mean first passage time. To assist visualization, the circles are colored based on the highest oligomer order the representing state contains. MSM trajectories of 10 ms based on the MSM transition probabilities can be found in Figure S8.
Figure 8
Figure 8
Allosteric modulation and GPCR oligomerization The activated GPCR generates an array of oligomeric assemblies, which couples to signaling partners, generating an array of supramolecular signaling complexes capable of initiating different signaling pathways. This mechanism of combinatory modulation of GPCR is responsive to the lipid bilayer environment and the conformational state of the receptor. Receptor activation, PIP2, and/or mini Gs binding, and an increase in receptor density in the bilayer all promote oligomerization.

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