Figure 1.
Protocol for ANM-restrained-MD simulations and β2AR conformations.
A. Structure of β2AR. Transmembrane helices 1–7 are labeled by numbers and colored in red, orange, yellow, green, blue, purple, and pink, respectively. Cytoplasmic helix 8 and the short extracellular helix below the binding cavity in extracellular loop 2 are colored in cyan. Ligand- and G-protein binding sites are shown by arrows. The palmitolyl group that is anchored to the membrane from the H8 is also shown in cyan. B. The protocol for generating the ensemble of conformations by ANM-restrained-MD algorithm. C. Ribbon diagrams of β2AR conformations. Front view (top) and back view (bottom) of β2AR conformers generated by ANM-restrained-MD are shown.
Figure 2.
Microdomains of β2AR stabilized by new interactions and water molecules.
A. Water molecules stabilizing the conserved NPXXY motif (left) and the Asn-Asp pair (right) within the transmembrane region, and (B) the critical catecholamine binding Ser203 and Ser207 residues at H5 in conformations where they both point to the ligand binding pocket and connected through a water molecule. C. The motion of extracellular loop two (EC2) and the residues that form the salt bridge at the EC site. The motion found by ANM-restrained-MD to break the salt bridge at the extracellular site and the opening of the extracellular site is shown. ANM-restrained-MD conformation and the carazolol-bound structures are in solid and transparent colors, respectively. The side chains of Asp192 at EC2 and Lys305 at H7 that form the salt bridge at the inactive state of β2AR are displayed on both structures. The motion of the EC2 including the short helix is depicted by red arrows.
Table 1.
β2AR residues that are shown to be interacting with agonists.
Figure 3.
RMSD profiles as a function of residue index for the ANM-restrained-MD conformations that accommodate salmeterol and epinephrine.
Red and blue curves represent RMSD per residue between the starting structure and the conformations that accommodate salmeterol and epinephrine, respectively. In both complexes, not only cytoplasmic region that binds to G-protein but also the extracellular region exhibits higher motilities. Although the overall RMSD profile of the extracellular region tends to maintain similar features in both conformations, 8M has larger structural rearrangements at EC2 and at the extracellular end of the helices that are connected by this loop. These structural arrangements lead to the accommodation of salmeterol at the extracellular region. The sequence ranges of the helices (H1–H8) are indicated by the labels on the upper abscissa and distinguished by gray bands. The color code of the helices that are used in the ribbon diagrams of β2AR are also displayed as colored bands at the bottom of the graph.
Figure 4.
Binding of salmeterol to β2AR.
A. Interactions of salmeterol at the binding site of an ANM-restrained-MD conformation; B. The location of salmeterol tail forming a ‘lid’ with the extracellular loop 2 (EC2) of β2AR from the extracellular site view. The flexible tail of salmeterol folds parallel to the EC2 and stabilizes the rest of the ligand forming a lid at the extracellular site. The green arrow shows the direction that the salmeterol tail runs similar to a beta-sheet structure. C. The residues lining the salmeterol binding pocket; D. The stabilization of the aromatic ring of salmeterol by Val114, Thr118 at H3, Ser203 and Ser207 at H5, and Phe290 atH6. Carbon, oxygen, nitrogen and hydrogen atoms of salmeterol is colored green, red, blue and white; respectively. ANM-restrained-MD conformation that is able to accommodate inside the ligand binding pocket is shown in ribbon diagram with transparent helices. The residues with the majority of atoms within the 3.5 Å making specific interactions with salmeterol are displayed. The EC2 that closely interacts with salmeterol is also show in green ribbon diagram.
Figure 5.
Binding of salmeterol to the active BI-167107 bound form of β2AR.
Salmeterol occupies the same location in the crystal structure of the active β2AR as we identified by ANM-restrained-MD for the conformation-salmeterol complex. The residues that have atoms in 3.5 Å are displayed and those that are not interacting with salmeterol in ANM-restrained-MD conformation are labeled in red. These are Trp286 at H6, Ile 309 at H7, and Phe194 at EC2. Carbon atoms of salmeterol are colored in pink. The rest of the atoms are colored the same as in Figure 3.
Figure 6.
The complexes of epinephrine with an ANM-restrained-MD conformation and the active crystal structure.
A. ANM-restrained-MD conformation-Epinephrine Complex. Epinephrine is located at the orthosteric binding site. The meta- and para- hydroxyl groups of the catechol ring are interacting with Ser203 and Ser204 at H5, respectively. Both β-hydroxyl and amine groups of epinephrine are forming hydrogen bonds with Asp113. The carbon atoms of epinephrine are colored green. B. BI-167107 bound active structure-Epinephrine Complex. The first pose of epinephrine in the BI-167107 bound active structure is located at the orthosteric binding pocket near the experimentally verified ligand binding residues active crystal structure. However, it is not forming any hydrogen bonds with any of these residues such as serines at H5 and Asn113 at H3.