Adrenaline-activated structure of Beta(2)-adrenoceptor stabi

Adrenaline-activated structure of Beta(2)-adrenoceptor stabilized by an engineered nanobody

Procedure

Results

Extensive polar network + small binding pocket = enhanced agonist affinity

Conclusion

Inactive conformation of thermostabilized Beta(1)-AR bound to isoprenaline

Active conformation of Beta(2)-AR bound to adrenaline

Creating Nb80 Mutants

Similarities

• Both structures show H-bond between Ser211
• B-hydroxylamine moiety engages the conserved residues Asp and Asn in a similar manner

Libraries of Nb80 mutants were constructed where residues at the receptor-binding surface were randomized with conservative substitutions.

The library was then subject to six rounds of selection to see if any nanobody mutants would create stronger affinity to low-affinity agonists like adrenaline.

• Developement of Nb6B9
• Revealed active-state structure of Beta(2)-AR with low affinity agonist, adrenaline
• Possible potentials for drug discovery

Yeast Surface Display

Selection Process

The researchers displayed Nb80 on the surface of a yeast strain

Nanobody 80 fuses to the N terminus of aga2p (a yeast cell wall protein) through covalent interactions

Detergent-solubilized biotinylated B2AR after pre-incubation of the receptor with the agonist BI167107 or inverse agonist carazolol stained and purified the yeast Nanobody80 complex

The library was subject to six rounds

1st the library was positively selected with decreasing concentrations of BI167107 bound B2AR.

Before positive selections of rounds 2-5, the library was negatively selected against binding to inverse-agonist occupied B2AR

This negative selection was done to remove variants that lost conformational specificity.

Selection Process

As the rounds of the selection process increased, the affinity of the agonist to the receptor with the help of the mutant nanobody caused the biotin molecule on the receptor to increase fluorescence

The carazolol has low fluorescence due to the fact that no agonist binds to the receptor resulting in the biotin not causing any high fluorescence.

The final round of selection, the scientists enriched the variants with the slowest dissociation rates.

Slow dissociation rates means a high affinity between the nanobody and the low binding affinity agonist in the B2AR receptor

• Limited network of polar contacts
• Extreme exposure to the extracellular solvent and isoprenaline displaced towards the extracellular side of the receptor

• Extended polar contact network linking orthosteric site to ECL2 and 3
• The catechol head group is completely enclosed within the binding site---results in contraction upon binding

This article presents and compares the active-state structure of beta(2)-AR bound to the three different agonists:

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Comparison of agonist-binding modes

The comparison of adrenaline with HBI shows a conserved agonist-binding mode

Despite the chemical diversity of the different ligands, the structures of B2AR bound to the agonists have very similar overall structures.

The comparison of BI167107 with HBI shows a conserved agonist-binding mode.

Due to this difference in hydrogen bonding between BI167107 and adrenaline, the Asn 293 and TM6 residues in the receptor shift inwards in the adrenaline-bound structure, leading to a cascade of changes

Comparison of agonist-binding modes

• a water molecule mediates a hydrogen bond between Tyr326 of the NPxxy motif and Tyr219 on the intracellular side of the transmembrane helix 5 (TM5)
• Water mediated h-bond contributes to the active-state stability of Beta(2)-AR

The comparison of BI167107 and adrenaline shows a conserved agonist-binding mode.

To maintain hydrogen bonds between Asn293 and the ring, the receptor undergoes a shift in the extracellular side of the TM6 leading to a conformational rearrangement in the extracellular loop.

Red dotted line is for BI167107 and blue dotted line is for adrenaline hydrogen bonding.

Comparison of agonist-binding modes

Intracellular side of the receptor

Beta(2)-adrenoceptor

• This figure shows similarity in the receptor conformation
• Water molecules resolved in the extracellular region of the receptor

1. ultra-high affinity agonist, BI167107

2. high affinity catecholamine agonist hydroxybenzyl isoproterenol

3. low affinity endogenous agonist Adrenaline

• natural agnist of beta(2)-AR
• Binds with low affinity to the beta(2)-AR
• Chemically unstable

Adrenaline

• nanobody used to obtain the first active-state structure of beta(2)-AR
• crystals obtained only with high affinity agonist - BI167107

Nb80

Nanobody

Changes to Nb80........

• Improve affinity of Nb80 to the agonist-bound receptor
• Minimize the conformational changes to the receptor after the nanobody binds
• conformational selectivity must be maintained

• Type of a G-protein-coupled receptor (GPCR)
• Integral membrane protein
• Binds ligand-molecules extracellular and produce cellular responses inside the cell
• Three types of ligands:
• Agonist- causes receptor stimulation
• Antagonist-no effect on receptor and blocks the effect of agonist
• Receptor interact with Stimulatory protein (Gs) or inhibitory Protein (Gi) inside the cell
• Importance: located throughout the body, important for cardiovascular and bronchial therapy

• conformationally selective single-domain camelid antibody fragment
• used to trap the actie-state structurs of the agonist-bound beta(2)-AR
• mimics Gs protein of GPCR

Nanobody 6B9

Nanobody 6B9 (Nb6B9)

This engineered nanobody shows a comparable increase in B2AR affinity for adrenaline in the presence of Nb6B9 compared to Gprotein Gs.

As talked about before, adrenaline has a low affinity when bound to the receptor. This new nanobody increases the affinity of this agonist with the help of the engeineered nanobody

From the screening process, Nb6B9 was chosen from the variants screened in the final round of selection.

This was due to the fact that it was one of the highest- affinity binders tested.

Compared to the wildtype nanobody, this engineered nanobody had a higher dissociation half-life and a 13-fold reduction in the dissociation rate.

References

1. Ring, A. M., A. Manglik, A. C. Kruse, D. M. Enos, I. W. Weis, C. K. Garcia, and K. B. Kobilka. "Adrenaline-activated Structure of β2-adrenoceptor Stabilized by an Engineered Nanobody." Nature 502.7472 (2013): 575-79. Print.

2. "Pharmtastic." A Review of the Development of Structure-specific Drugs for Beta-­2 Adrenergic Receptors. N.p., n.d. Web. 13 Jan. 2014.