A total of approximately 100 crystals was obtained, of which the 13 best yielded diffraction data good enough for a 2.6-? Tofacitinib baldness resolution. In Fig. 1A, a representation of the receptor structure (without T4L) is shown, with a closer look at the ligand binding site in Fig. 1B. The final refined structure thus refers to a fusion protein of the adenosine A2A receptor with T4L (including amino acid residues Ile3 to Gln310 of the receptor and residues 2 to 161 of T4L), a number of lipids modeled as stearic acid molecules, sulfate ions, ordered water molecules, and the antagonist ZM241385. At some parts of the polypeptide chain, the weak electron density did not allow a precise structure determination. This was true for the first two amino acids of the receptor (Met1�CPro2), amino acids Glu311 to Ala316 in the carboxyl tail, and the tip of the second extracellular loop (Gln148�CSer156).
Unlike the two ��-adrenergic receptors, the A2A receptor structure did not provide evidence for the presence of cholesterol molecules. Fig. 1. Left, crystal structure of the adenosine A2A receptor. Brown, ��-helical elements; dark blue, ZM241385; yellow, disulfide bridges; pink, extracellular domain; red, stearic acid molecules; and light blue, intracellular domain. Membrane boundaries … C. Pharmacological Characterization Such a highly engineered receptor construct necessitated a thorough pharmacological characterization with respect to signaling and ligand binding properties. Signaling, measured as modulation of cAMP production, was completely abrogated, most probably because of the insertion of T4L in the third intracellular loop of the receptor.
Compared with the wild-type receptor, the construct displayed virtually identical affinity for the antagonist ZM241385 in radioligand binding studies, whereas agonist affinity was somewhat higher than observed for the wild-type construct. High sodium chloride concentrations, essential for the generation of crystals, did not affect antagonist affinity, whereas agonist affinity was reduced to a similar value for wild-type and engineered receptor, in line with earlier observations (Gao and IJzerman, 2000). This suggests that the antagonist-binding site in the crystal structure was not affected by the substantial modifications to the receptor protein. D. Structural Characteristics 1. The Ligand Binding Site.
The A2A receptor structure is quite different from the other GPCRs for which crystal structures are available. First, ZM241385 binds in an extended conformation protruding into the extracellular domain, whereas the ligand-binding site of ��-receptor antagonists seems to reside within the transmembrane domain. Therefore, the amino acids interacting with the ligand are found mainly on helices 3, 5, 6, and 7 in the transmembrane domain as well Dacomitinib as in extracellular loops 2 and 3 (see also Fig. 1B).