Ligand/Receptor Complex-Assembling

For ligand docking of AgRP into the inactive MC4R model (see section Ligand models) the known structural AgRP fragment (see section Ligand models) was placed manually in the extracellular solvent phase at approximately 5 Å separation from the hMC4R surface in close spatial proximity above the extracellular loops. A known interaction between the amino acids of the ligand and the receptor involved in binding (16, 63–66) was used as a distance constraint. In particular, the specific AgRP motif ₁₁₁RFFN₁₁₄ is essential for interaction with the orthosteric site of the MC4R and the MC3R (67, 68). In accordance to others [reviewed in Ericson et al. (9)], we thereby assumed that the positively charged side chain of R111 (AgRP) interacts with the negatively charged side chains of D122 and D126 (MC4R). Molecular dynamic simulations (300 K, 3 ns) were initiated with a distant constraint of 2 Å between the side chains of AgRP R111 and hMC4R D126. All backbone atoms of the receptor helices and the ligand were constrained. The resulting model was energetically minimized, followed by a second dynamic simulation (2 ns) without any distance constraints on the ligand backbone. The resulting complex model was energetically minimized without any constraint.