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Membrane Mimetic-Dependence of GPCR Energy Landscapes.

ABSTRACT: Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature ¹⁹F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A_2A adenosine receptor (A_2AAR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A_2AAR in lipid nanodiscs were markedly dependent on the efficacy of bound drugs. While antagonist complexes displayed only modest changes as the temperature rose, both full and partial agonist complexes exhibited substantial increases in the active state population. Importantly, the temperature-dependent response of complexes with both full and partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. In striking contrast to observations within lipid nanodiscs, in detergent micelles the active state population exhibited different behavior for A_2AAR complexes with both full and partial agonists. This underscores the importance of the protein environment in understanding the thermodynamics of GPCR activation.

SUBMITTER: Thakur N

PROVIDER: S-EPMC10614885 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Membrane Mimetic-Dependence of GPCR Energy Landscapes.

Thakur Naveen N Ray Arka Prabha AP Lyman Edward E Gao Zhan-Guo ZG Jacobson Kenneth A KA Eddy Matthew T MT

bioRxiv : the preprint server for biology 20231019

Protein function strongly depends on temperature, which is related to temperature-dependent changes in the equilibria of protein conformational states. We leveraged variable-temperature 19F-NMR spectroscopy to interrogate the temperature dependence of the conformational landscape of the human A2A adenosine receptor (A2AAR), a class A GPCR. Temperature-induced changes in the conformational equilibria of A2AAR in lipid nanodiscs were markedly dependent o ...[more]

PMID: 37905159

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Project description:Membrane reorientation of oncogenic RAS proteins is emerging as an important modulator of their functions. Previous studies have shown that the most common orientations include those with either the three C-terminal α-helices (OS1) or N-terminal β-strands (OS2) of the catalytic domain facing the membrane. OS1 and OS2 differ by the degree to which the effector-interacting surface is occluded by the membrane. However, the relative stability of these states and the rates of transition between them remained undetermined. How mutations might modulate preferences for specific orientation states is also far from clear. The current work attempted to address these questions through a comprehensive analysis of two 20 μs-long atomistic molecular dynamics simulations. The simulations were conducted on the oncogenic G12D and Q61H KRAS mutants bound to an anionic lipid bilayer. G12D and Q61H are among the most prevalent cancer-causing mutations at the P-loop and switch 2 regions of KRAS, respectively. We found that both mutants fluctuate in a similar manner between OS1 and OS2 via an intermediate orientation OS0, and both favor the signaling competent OS1 and OS0 over the occluded OS2. However, they differ in the details, such as in the extent to which they sample OS1. Analysis of the orientation free-energy landscapes estimated from the simulations indicate that OS1 and OS2 are the most stable states. However, the overall free energy surface is rugged, indicating a large diversity of conformations including at least two substates in each orientation state that differ in stability only by about 0.5-1.0 kcal/mol. Reversible transitions between OS1 and OS2 occur via two well-defined pathways that traverse OS0. In the minimum energy path, helix 4 remains close to the membrane as the angle of the catalytic domain from the membrane plane changes, resulting in a barrier of ∼1 kcal/mol for OS1/OS2 interconversions. Estimation of the rates of the various transitions based on survival probabilities yielded two rate constants in the order of 107 and 106 s-1, which we attribute to intrinsic protein conformational dynamics and transient protein-lipid interactions, respectively. The faster process dominates every transition, confirming a previous suggestion that RAS membrane reorientation is driven by conformational fluctuations rather than protein-lipid interactions.

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Membrane Mimetic-Dependence of GPCR Energy Landscapes.

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Membrane Mimetic-Dependence of GPCR Energy Landscapes.

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