Prof. Brian Kobilka’s group reported novel mechanism of β2AR regulation by an intracellular positive allosteric modulator
On June 28th, 2019, Prof. Brian Kobilka’s group in the School of Medicine, Tsinghua University, in collaboration with Professor Robert Lefkowitz’s group at Duke University, published a paper in Science, entitled “Mechanism of β2AR regulation by an intracellular positive allosteric modulator”. The paper reported a crystal structure of the active state β2AR in complex with orthosteric agonist BI167107, G protein mimic nanobody Nb6B9 and a newly identified, intracellular positive allosteric modulator Cmpd-6FA. The work identified a novel mechanism for allosteric modulation of a G protein coupled receptor (GPCR).
GPCRs are the subjects of intense drug discovery efforts. Orthosteric drugs, which target the natural hormone binding site of GPCRs, are the most common therapeutics. However orthosteric drugs often have problems with subtype selectivity, as orthosteric binding pockets are highly conserved among GPCRs of the same subfamily, for examples the nine adrenergic receptors in human. Allosteric drugs are more likely to be subtype selective because they bind to less conserved regions. Recently, many GPCR structures bound with a variety of allosteric modulators have been reported. Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive state. Before the publication of this paper, only one active GPCR bound to a small molecule positive allosteric modulator (PAM) has been reported, namely, the structure of M2 muscarinic acetylcholine receptor bound with LY2119620, which was solved by Brian Kobilka’s research group at Stanford in 2013 (Kruse et al., 2013).
The Beta2 adrenergic receptor (β2AR) belongs to the GPCR family and plays essential roles in cardiovascular and respiratory physiology. Orthosteric agonists for the β2AR are commonly used to treat asthma and chronic obstructive lung disease. In 2017, Prof. Brian Kobilka’s group at Tsinghua University and Prof. Robert Lefkowitz’s group at Duke University jointly reported the first crystal structure of the β2AR bound to an intracellular negative allosteric modulator Cmpd-15(Liu et al., 2017). Recently, Prof. Robert Lefkowitz’s group discovered Cmpd-6 as a positive allosteric modulator for the β2AR through screening DNA encoded libraries (Ahn et al., 2018). Cmpd-6 exhibits robust positive cooperativity with orthosteric agonists and transducers like G protein and arrestin.
Prof. Brian Kobilka’s research group and Prof. Robert Lefkowitz’s research group established a collaboration to determine the complex structure of the active state β2AR bound with Cmpd-6. However the initial trials failed, no electron density was observed for Cmpd-6. The researchers reasoned that it was due to Cmpd-6’s micromolar affinity and poor solubility. Prof. Robert Lefkowitz’s group further optimized the compound by adding a free carboxylic acid group to its terminal amide site (previously used for conjugating its DNA tag) to get an analog (Cmpd-6FA) with better solubility. Dr. Xiangyu Liu and colleagues in Prof. Brian Kobilka’s group found that the compounds were more soluble in solutions with high concentration of PEG400 and successfully crystallized active state β2AR and solved the structure at a resolution of 3.2 A. Cmpd-6FA bound to a pocket formed by transmembrane (TM) helices 2, 3, 4 and intracellular loop 2 (ICL2). (Figure 1).
Figure 1: The chemical structure of Cmpd-6FA and the crystal structure of active state β2AR bound with orthosteric agonist BI167107, G protein mimic nanobody Nb6B9 and allosteric agonist Cmpd-6FA.
Interestingly, ICL2 is a random coil in the inactive-state β2AR and is a two-turn α-helix in active-state β2AR. Cmpd-6FA binding stabilized the α-helical conformation of ICL2 which facilitates β2AR-Gs protein coupling. In addition, the formation of α-helix in ICL2 requires inward movement of TM3, which in turn dictates an outward movement of TM5 and TM6. The outward movement of TM5 and TM6 is a hallmark of GPCR activation. In this way, Cmpd-6FA binding increases of population of receptors adopting active conformations, which have higher affinity for agonists (Figure 2). This mode of action differs from the mechanism previously reported for the PAM LY2119620 that targets M2 muscarinic acetylcholine receptor; whereby the allosteric modulator binds within the extracellular vestibule and directly prevents agonist dissociation (Kruse et al., 2013).
Figure 2: Mechanism of allosteric activation of the β2AR by Cmpd-6FA. (A), ,Cmpd-6FA binding facilitates β2AR-Gs coupling. (B), Cmpd-6FA binding requires inward movement of TM3, which in turn induces outward movement of TM5 and TM6. As a result, Cmpd-6FA binding increases the population of receptors adopting active conformations.
The structure model was further supported by a structure-activity relationship study using a set of Cmpd-6 derivatives and by a gain-of-function mutagenesis study on the β1AR. Cmpd-6 is subtype selective towards the β2AR. It is much more efficacious in enhancing agonist binding for the β2AR than the closely related β1AR. Sequence alignment of the β2AR and β1AR reveals that 7 out of the 14 amino acid residues that form the Cmpd-6FA allosteric site are not conserved. When these 7 residues of the β1AR were mutated to match their counterparts in the β2AR, agonist affinity was enhanced by 13-fold in the presence of Cmpd-6.
The Cmpd-6FA binding pocket may represent a general locale for positive allostery in class A GPCRs. It is partially overlapped with the allosteric agonist AP8 binding pocket on the free fatty acid receptor GPR40(Lu et al., 2017). And recently, mutagenesis studies suggested EDTQ, a PAM for the dopamine D1 receptor, binds to a site near ICL2(Wang et al., 2018). The β2AR-Cmpd-6FA structure may serve as a template to guide allosteric agonist development for other GPCRs.
This work is the result of a collaboration between Professor Brian Kobilka’s lab at Tsinghua University and Professor Robert Lefkowitz’s lab at Duke University. Professor Brian Kobilka and Professor Robert Lefkowitz are corresponding authors of the paper. Dr. Xiangyu Liu from School of Medicine, Tsinghua University, Dr. Ali Masoudi and Dr. Alem W. Kahsai from Duke University Medical Center are co-first authors of the paper. The work is supported by grants from Beijing Advanced Innovation Center for Structural Biology and Tsinghua-Peking Joint Center for Life Sciences. Crystal diffraction and data collection was supported by Spring-8 synchrotron radiation facility, Japan.
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References:
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Source: School of Medicine
Editor: Guo Lili
