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


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

Adernergic Synapse
Adernergic Synapse


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
Norephrine
Norephrine
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.


B₂AR Active

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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.

When B2AR is activated, the G-protein disassociates. A surrogate nanobody, , that is a camelid antibody produced to mimic the G-protein needed for an active state of the B2AR.

Active B₂AR in complex with BI-167107: and Interactions


Ligands

To understand activation of β₂AR 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 reduces the activity to the level below that of 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 for their β₂AR blocking activity, they are frequently prescribed for their action on the Beta-1 Adrenergic Receptors in people with cardiovascular disorders such as hypertension and angina. A number of selective β₂AR agonists are used for the treatment of asthma and chronic obstructive pulmonary disease (COPD).


Name Description Chemical Structure Photo
Isoproterenol Isoproterenol is an agonist that is structurally similar to NE and readily binds to β₂AR with high affinity. Isoproterenol contains an isopropyl amino group and a catechol group.
BI-167107 The active state of β₂AR 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 β₂AR. Carazolol contains a propylamino and a carbazole group.


Comparison of the Inverse Agonist and the Agonist

Notable differences between carazolol and both isoproterenol and the natural agonist norepinephrine are that: (i) Carazolol lacks the hydroxyl groups thought to be necessary for the activation of β₂AR. (ii)The side chain of carazolol is two atoms (one carbon and one oxygen) longer in length from the amino group to the carbazole moiety. These are common characteristics of β₂AR antagonists.


The binding mode of isoproterenol and carazolol in B2AR. Hydrophobic residues are displayed in yellow. Polar interactions are displayed with residues in cyan, oxygen in red, and hydrogen in white. (a) A model of B2AR in its active state in complex with isoproterenol.  (b) B2AR in its inactive state in complex with carazolol.
The binding mode of isoproterenol and carazolol in B2AR. Hydrophobic residues are displayed in yellow. Polar interactions are displayed with residues in cyan, oxygen in red, and hydrogen in white. (a) A model of B2AR in its active state in complex with isoproterenol. (b) B2AR in its inactive state in complex with carazolol.

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

Conformational Changes in B2AR from Inactive State(2rh1) to Active State(3p0g)

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When an agonist is in the 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.

Models of  isoproternol binding to two B2AR structures. (a) Inactive B2AR: 4.78Å distance between the catechol-OH of the ligand and Ser207 of TM5  is too large for a H-bond. (b)  Active B2AR: A hydrogen bond distance of 2.17Å  between the catechol-OH of the ligand and Ser207  on TM5 is shown.
Models of isoproternol binding to two B2AR structures. (a) Inactive B2AR: 4.78Å distance between the catechol-OH of the ligand and Ser207 of TM5 is too large for a H-bond. (b) Active B2AR: A hydrogen bond distance of 2.17Å between the catechol-OH of the ligand and Ser207 on TM5 is shown.
Model of carazolol binding to B2AR structure. (a) Active B2AR: there is a steric clash between the ligand and Ser207 of TM5. (b)Inactive B2AR: carazolol in B2AR fits perfectly and blocks the agonist from entering  the binding pocket.
Model of carazolol binding to B2AR structure. (a) Active B2AR: there is a steric clash between the ligand and Ser207 of TM5. (b)Inactive B2AR: carazolol in B2AR fits perfectly and blocks the agonist from entering the binding pocket.

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.

Molecular Morph

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.


Reference

1. Vadim Cherezov, Daniel M. Rosenbaum, Michael A. Hanson, Søren G. F. Rasmussen, Foon Sun Thian, Tong Sun Kobilka, Hee-Jung Choi, Peter Kuhn, William I. Weis, Brian K. Kobilka, Raymond C. Stevens (2007). High Resolution Crystal Structure of an Engineered Human B2-Adrenergic G Protein- Coupled Receptor Science 318, 1258-1265.

2.Søren G. F. Rasmussen, Hee-Jung Choi, Juan Jose Fung, Els Pardon, Paola Casarosa, Pil Seok Chae, Brian T. DeVree, Daniel M. Rosenbaum, Foon Sun Thian, Tong Sun Kobilka, Andreas Schnapp, Ingo Konetzki, Roger K. Sunahara,Samuel H. Gellman, Alexander Pautsch, Jan Steyaert, William I. Weis & Brian K. Kobilka (2011). Structure of a nanobody-stabilized active state of the B2 adrenoceptor Nature 469, 175-180.


Acknowledgements

Camille and Henry Dreyfus Foundation, The Rockefeller University Center for Clinical and Translational Science, The Rockefeller University Science Outreach Program, Howard Hughes Medical Institute Pre-college Program, Center for BioMolecular Modeling, Milwaukee School of Engineering, The David A. Cofrin Center for Biomedical Information, in the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College,


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