Abstract
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-016-0061-9) contains supplementary material, which is available to authorized users.
Highlights
This 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
As the first hormone-activated G protein coupled receptors (GPCRs) structure to be reported by X-ray crystallography [1, 2], the high resolution structural information was obtained through the elimination of the third intracellular loop (ICL3) replaced with the protein T4 lysozyme (T4L) and the C-terminal tail in order to increase both the proteolytic stability and crystallizability
hydrogen/deuterium exchange (HDX) data suggested ICL3’s role as a molecular switch, where antagonist and inverse agonist binding shifted the equilibrium toward inactive states, which is characterized by protection to exchange in the ICL3 loop and flanking regions of transmembrane helix (TM) helices V and VI
Summary
This 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. 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). HDX data suggested ICL3’s role as a molecular switch, where antagonist and inverse agonist binding shifted the equilibrium toward inactive states, which is characterized by protection to exchange (i.e., stabilization) in the ICL3 loop and flanking regions of TM helices V and VI. During the 1 μs long MD run, the loop model found a very inactive conformation towards 600 ns, when TM6 moved towards the receptor’s core region with ICL3 packing underneath the membrane and blocking the G-protein’s binding site. ICL3 preserved its closed conformation and the receptor’s overall dynamics has decreased significantly with only minor fluctuations for the remaining 400 ns, and became similar to the clipped model’s dynamics, which showed no major variations
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have