Investigation of allosteric coupling in human β2-adrenergic receptor in the presence of intracellular loop 3

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.

Signaling within Allosteric Machines: Signal Transmission Pathways Inside G Protein-Coupled Receptors

“…Another involvement of TM5 and TM6 was also recently investigated by Ozgur et al [ 56 ]. They computationally studied the β 2 adrenergic receptor together with the intracellular loop 3 (ICL3), which is known to be problematic and frequently replaced by more stable protein fragments in X-ray crystallography studies in order to facilitate crystallization.…”

Structural insights into positive and negative allosteric regulation of a G protein-coupled receptor through protein-lipid interactions

“…However, these observations may be influenced by the absence of ICL3 (which connects TM5 and TM6) in some of these MD simulations. Indeed, in recent MD studies which did include ICL3, receptor behaviour was found to be altered because of noticeable allosteric effects 23 , 25 . Likewise, experimental data suggests ICL3 is important for the spontaneous activation of β2AR 43 .…”

Ligand‐binding affinity of alternative conformers of human β2‐adrenergic receptor in the presence of intracellular loop 3 (ICL3) and their potential use in virtual screening studies

“…Figure 1c illustrates this change by superimposing the alternative inactive state over the inactive X-ray crystal structure (PDB id: 2RH1). Previously, a simulation study on human β 2 -AR by Ozgur et al (2016) was conducted using several bond restraints at the ligand-binding site in order to investigate the allosteric coupling between the intra-and extracellular regions of the receptor. Our last simulation was conducted using the final snapshot of that restrained run as the initial frame, with all restraints removed, in order to observe the conformational change in ICL3, which was initially found in a packed state.…”

“…However, neither active nor inactive crystal states captured the full flexibility of the receptor, which presents a wide range of conformers that have distinct atomic‐level details in the ligand‐binding pocket (Kobilka & Deupi, ). Furthermore, ICL3 connects the cytoplasmic ends of transmembrane helices V (TM5) and VI (TM6) and has a functional role for both the recognition and the activation of G proteins (Ozgur, Doruker, & Akten, ; Rasmussen et al., ). Unlike other intracellular loop regions, ICL3 has a highly variable length and sequence among the members of the GPCR superfamily; thus, it is believed that a GPCR's selectivity to different G proteins originated in the structural uniqueness of ICL3 (Ikezu, Okamoto, Ogata, & Nishimot, ).…”