Reanalysis of a [mu] opioid receptor crystal structure reveals a covalent adduct with BU72

The first crystal structure of the active [mu] opioid receptor ([mu]OR) exhibited several unexplained features. The ligand BU72 exhibited many extreme deviations from ideal geometry, along with unexplained electron density. I previously showed that inverting the benzylic configuration resolved these problems, establishing revised stereochemistry of BU72 and its analog BU74. However, another problem remains unresolved: additional unexplained electron density contacts both BU72 and a histidine residue in the N-terminus, revealing the presence of an as-yet unidentified atom. These short contacts and uninterrupted density are inconsistent with non-covalent interactions. Therefore, BU72 and [mu]OR form a covalent adduct, rather than representing two separate entities as in the original model. A subsequently proposed magnesium complex is inconsistent with multiple lines of evidence. However, oxygen fits the unexplained density well. While the structure I propose is tentative, similar adducts have been reported previously in the presence of reactive oxygen species. Moreover, known sources of reactive oxygen species were present: HEPES buffer, nickel ions, and a sequence motif that forms redox-active nickel complexes. This motif contacts the unexplained density. The adduct exhibits severe strain, and the tethered N-terminus forms contacts with adjacent residues. These forces, along with the nanobody used as a G protein substitute, would be expected to influence the receptor conformation. Consistent with this, the intracellular end of the structure differs markedly from subsequent structures of active [mu]OR bound to G.sub.i protein. Later G.sub.i-bound structures are likely to be more accurate templates for ligand docking and modelling of active G protein-bound [mu]OR. The possibility of reactions like this should be considered in the choice of protein truncation sites and purification conditions, and in the interpretation of excess or unexplained density.