Dataset Information

Cellular Lipids Regulate the Conformational Ensembles of the Disordered Intracellular Loop 3 in β2 Adrenergic Receptor.

ABSTRACT: The structurally disordered intracellular loops (ICLs) of G protein-coupled receptors (GPCRs) play a critical role in G protein coupling. In our previous work, we used a combination of FRET-based and computational methodologies to show that the third intracellular loop (ICL3) modulates the activity and G protein coupling selectivity in GPCRs. In the current study, we have uncovered the role of several lipid components in modulating the conformational ensemble of ICL3 of the β2-adrenergic receptor (β2AR). Our findings indicate that phosphatidylinositol 4,5-bisphosphate (PIP2) in the inner leaflet of the membrane bilayer acts as a stabilizing anchor for ICL3, opening the intracellular cavity to facilitate G protein coupling. This interaction between PIP2 and ICL3 causes tilting of β2AR within the cellular membrane. Notably, this tilting of the receptor is supported by ganglioside GM3 stabilizing the extracellular loops on the outer leaflet of the bilayer, thereby exerting an allosteric effect on the orthosteric ligand binding pocket. Our results underscore the significance of lipids in modulating GPCR activity, proposing an allosteric mechanism that occurs through the receptor's orientation within the membrane.

SUBMITTER: Mukhaleva E

PROVIDER: S-EPMC10705491 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Cellular Lipids Regulate the Conformational Ensembles of the Disordered Intracellular Loop 3 in β2 Adrenergic Receptor.

Mukhaleva Elizaveta E Yang Tianyi T Sadler Fredrik F Sivaramakrishnan Sivaraj S Ma Ning N Vaidehi Nagarajan N

bioRxiv : the preprint server for biology 20231129

The structurally disordered intracellular loops (ICLs) of G protein-coupled receptors (GPCRs) play a critical role in G protein coupling. In our previous work, we used a combination of FRET-based and computational methodologies to show that the third intracellular loop (ICL3) modulates the activity and G protein coupling selectivity in GPCRs. In the current study, we have uncovered the role of several lipid components in modulating the conformational ensemble of ICL3 of the β2-adrenergic recepto ...[more]

PMID: 38077083

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Project description:BackgroundThis study investigates the allosteric coupling that exists between the intra- and extracellular parts of human β2-adrenergic receptor (β2-AR), in the presence of the intracellular loop 3 (ICL3), which is missing in all crystallographic experiments and most of the simulation studies reported so far. Our recent 1 μs long MD run has revealed a transition to the so-called very inactive state of the receptor, in which ICL3 packed under the G protein's binding cavity and completely blocked its accessibility to G protein. Simultaneously, an outward tilt of transmembrane helix 5 (TM5) caused an expansion of the extracellular ligand-binding site. In the current study, we performed independent runs with a total duration of 4 μs to further investigate the very inactive state with packed ICL3 and the allosteric coupling event (three unrestrained runs and five runs with bond restraints at the ligand-binding site).ResultsIn all three independent unrestrained runs (each 500 ns long), ICL3 preserved its initially packed/closed conformation within the studied time frame, suggesting an inhibition of the receptor's activity. Specific bond restraints were later imposed between some key residues at the ligand-binding site, which have been experimentally determined to interact with the ligand. Restraining the binding site region to an open state facilitated ICL3 closure, whereas a relatively constrained/closed binding site hindered ICL3 packing. However, the reverse operation, i.e. opening of the packed ICL3, could not be realized by restraining the binding site region to a closed state. Thus, any attempt failed to free the ICL3 from its locked state due to the presence of persistent hydrogen bonds.ConclusionsOverall, our simulations indicated that starting with very inactive states, the receptor stayed almost irreversibly inhibited, which in turn decreased the overall mobility of the receptor. Bond restraints which represented the geometric restrictions caused by ligands of various sizes when bound at the ligand-binding site, induced the expected conformational changes in TM5, TM6 and consequently, ICL3. Still, once ICL3 was packed, the allosteric coupling became ineffective due to strong hydrogen bonds connecting ICL3 to the core of the receptor.

Project description:We report a conformational switch between two distinct intrinsically disordered subensembles within the active site of a transcription factor. This switch highlights an evolutionary benefit conferred by the high plasticity of intrinsically disordered domains, namely, their potential to dynamically sample a heterogeneous conformational space housing multiple states with tailored properties. We focus on proto-oncogenic basic-helix-loop-helix (bHLH)-type transcription factors, as these play key roles in cell regulation and function. Despite intense research efforts, the understanding of structure-function relations of these transcription factors remains incomplete as they feature intrinsically disordered DNA-interaction domains that are difficult to characterize, theoretically as well as experimentally. Here we characterize the structural dynamics of the intrinsically disordered region DNA-binding site of the vital MYC-associated transcription factor X (MAX). Integrating nuclear magnetic resonance (NMR) measurements, molecular dynamics (MD) simulations, and electron paramagnetic resonance (EPR) measurements, we show that, in the absence of DNA, the binding site of the free MAX2 homodimer samples two intrinsically disordered conformational subensembles. These feature distinct structural properties: one subensemble consists of a set of highly flexible and spatially extended conformers, while the second features a set of "hinged" conformations. In this latter ensemble, the disordered N-terminal tails of MAX2 fold back along the dimer, forming transient long-range contacts with the HLH-region and thereby exposing the DNA binding site to the solvent. The features of these divergent substates suggest two mechanisms by which protein conformational dynamics in MAX2 might modulate DNA-complex formation: by enhanced initial recruitment of free DNA ligands, as a result of the wider conformational space sampled by the extended ensemble, and by direct exposure of the binding site and the corresponding strong electrostatic attractions presented while in the hinged conformations.

Dataset Information

Cellular Lipids Regulate the Conformational Ensembles of the Disordered Intracellular Loop 3 in β2 Adrenergic Receptor.

Publications

Cellular Lipids Regulate the Conformational Ensembles of the Disordered Intracellular Loop 3 in β2 Adrenergic Receptor.

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