The BU72-μ opioid receptor crystal structure is a covalent adduct

In the crystal structure of BU72 bound to the μ opioid receptor (μOR), the opioid clashes with an adjacent residue in the N-terminus; strong and unexplained electron density connects the two, centered on a point ~1.6 Å from each. This is too short for non-covalent interactions, implying covalent bonds to an unmodeled non-hydrogen atom. A magnesium ion has recently been proposed as a candidate. However, this would require unrealistically short bonds and an incomplete coordination shell. Moreover, the crystals were prepared without magnesium salts, but with components that can generate reactive oxygen species (ROS): HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fits the unexplained density well, giving a type of covalent adduct known to form in the presence of ROS, with reasonable geometry and no clashes. While the precise structure is tentative, the observed density firmly establishes covalent bonds linking ligand and residue. Severe strain is evident in the ligand, the tethered N-terminus, and the connecting bonds. This strain, along with interactions between the N-terminus and surrounding residues, is likely to distort the receptor conformation. The subsequent μOR-Gi structure, which differs in several features associated with activation, is therefore likely to be a more accurate model of the active receptor. The possibility of reactions like this should be considered in the choice of protein truncation sites and purification conditions.