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Modeling of Beta 2 Adrenergic Receptor: Ligand Binding and Activation

Students: Mary Acheampong, Kavita Bhikhi, Daviana Dueno, Bobby Glover, Lachoy Harris, Alafia Henry, Randol Mata, and Marisa Vanbrakle, Hostos-Lincoln Academy of Science

Teacher: Allison Granberry, Hostos-Lincoln Academy of Science

Mentors: Haregewein Assefa,Touro College of Pharmacy, Thijs Beuming, Schrodinger

Introduction

The Beta-2 Adrenergic Receptor (B₂AR) is a G-protein coupled receptor (GPCR) which, when stimulated by a catecholamine, causes the relaxation of various smooth muscles, and the production of glucose by glycogenolysis and gluconeogenesis. Pharmaceuticals acting through B2AR are important for treating asthma, chronic obstructive pulmonary disease (COPD), and premature labor. The structure of B2AR consists of 7-transmembrane domains, connected by three extracellular loops and three intracellular loops. At the base of the extracellular loops, buried within the transmembrane helices, there is a predominately hydrophobic binding pocket with several crucial polar residues that interact with ligands. Interestingly, certain polar interactions appear to play a role in the conversion of the receptor from an active to an inactive state. Recent crystallography of B2AR has revealed that the active state, relative to the inactive state, shows only minor changes in the binding pocket, whereas critical shifts occur at the cytoplasmic face. These conformational changes lead to a dissociation of the G-protein from the receptor, which then initiates a signaling cascade. The Hostos-Lincoln Academy SMART team (Students Modeling A Research Topic) modeled ligands in complex with B2AR using 3D printing technology. Supported by grants from the HHMI Precollege Program and the Camille and Henry Dreyfus Foundation.

Background Information

Adrenergic receptors are involved in activation of the sympathetic nervous system following sudden external stimuli.

After arrival of a nerve impulse, the neurotransmitter norepinephrine (NE) is released from the presynaptic terminal of the sympathetic neuron. NE is a tyrosine derived catecholamine containing an amino-hydroxyethyl and a catechol group.

NE binds to adrenergic receptors embedded in the postsynaptic effector cell membrane.

Following binding of NE, to either alpha or beta receptors, conformational changes in the receptor lead to a disassociation of the G protein from the cytoplasmic face of the receptor which activates a second messenger, initiating a signaling cascade.

Structure of Beta 2 Adrenergic Receptor

B2AR is a single chain that crosses the lipid membrane 7 times from the extracellular to cytoplasmic surface. There are 3 extracellular loops and 3 intracellular loops. The is located to the center of the extracellular surface.

Ligands

To understand activation of B₂AR by various ligands, it is first essential to define the term baseline activity. In the absence of a ligand within the binding pocket, there is some basal activity between the receptor and its signaling pathway. This activity is not considered an active state of the receptor but simply the baseline activity of the receptor. There are two extremes to the activity of the receptor that can be seen with binding of either an inverse agonist or an agonist. The inverse agonist completely stops the basal activity of the receptor whereas the agonist activates the receptor to its maximum. A ligand that is an antagonist actually has no effect on the basal activity of the receptor. An antagonist simply sterically blocks the receptor so that no other ligand can bind. Their activity would be considered baseline. Antagonists for the adrenergic receptors are commonly called Beta Blockers. Although Beta Blockers are not prescribed for B₂AR, they are frequently prescribed for Beta-1 Adrenergic Receptors in people with heart conditions.

Isoproterenol

Isoproterenol is an agonist that is structurally similar to NE and readily binds to B2AR with high affinity.

Isoproterenol contains an isopropyl amine group and a catechol group.

BI-167107

The active state of B2AR was crystallized using BI- 1671071 . Although it is not a catecholamine, it is a full agonist.

Carazolol

Carazolol is an inverse agonist designed to inhibit B₂AR.

Carazolol contains a propylamine and a carbazole group.

Comparison of the Inverse Agonist and the Agonist

Notable differences between carazolol and both isoproterenol and the natural agonist are that:

(i) Carazolol lacks the hydroxyl groups thought necessary for the activation of B₂AR.

(ii) Carazolol is one carbon longer in length from the amine group to the carbazole.

These are common characteristics of B₂AR antagonists.

Ligand Binding

Ligands share several key interactions in the binding pocket including:

(i)Polar interactions between:

The amine and Asp113 in TM3, Asn312 in TM7, and Tyr316 in TM7.

Hydroxyls and other h-bond donors and Ser207 in TM5, Ser203 in TM5, and Asn293 in TM6.

(ii) Hydrophobic interaction between ligand and Val117 in TM3, Phe193 in ECL2, Phe289 in TM6, and Phe290 inTM6.

Conformational Change

When an agonist is in the binding pocket a 2.1Å inward movement of TM5 at Ser207 is observed. This bulge at ser207 allows for a hydrogen bond between the ligand and the receptor. This interaction appears to be a key event in activation. After the agonist binds, there is a rearrangement of interactions between residues located beneath the binding pocket that contributes to a rotation and outward movement of TM6 at Phe282. This change is associated with the breaking of the ionic lock between Glu268 in TM6 and Arg131 in TM3, resulting in an 11.4Å outward movement of the helix at the cytoplasmic face. The coordinates for molecular morphs between inactive state of B2AR (2rh1) and active state (3p0g) were generated using iPyMOL and eMovie (http://www.weizmann.ac.il/ISPC/eMovie.html). Morphs, a series of 10 linear interpolations between a starting and finishing model, are useful when viewing the transition of a conformational change. This model of B2AR using morphs should not be thought of as precise animation of conformational changes upon activation but rather as a comparison of the inactive state to the active state.