scholarly journals The BU72-μ Opioid Receptor Crystal Structure Is a Covalent Adduct

2020 ◽  
Author(s):  
Thomas Anthony Munro
Keyword(s):  
Crystal Structure ◽  
Opioid Receptor ◽  
Coordination Shell ◽  
Oxygen Species ◽  
Nickel Ions ◽  
Redox Active ◽  
Covalent Adduct ◽  
N Terminus ◽  
Μ Opioid Receptor ◽  
Magnesium Salts

<div>In the crystal structure of BU72 bound to the μ opioid receptor, the opioid clashes with an adjacent residue, and unexplained electron density connects the two. It has been reported that this density can be filled by a magnesium ion. However, this proposal requires 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: HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fills the unexplained density, giving a known type of covalent adduct with reasonable geometry and no clashes. Strain is evident, but is consistent with tension from the tethered N-terminus.</div>

scholarly journals The BU72-μ Opioid Receptor Crystal Structure Is a Covalent Adduct

2020 ◽  
Author(s):  
Thomas Anthony Munro
Keyword(s):  
Crystal Structure ◽  
Opioid Receptor ◽  
Coordination Shell ◽  
Oxygen Species ◽  
Nickel Ions ◽  
Redox Active ◽  
Covalent Adduct ◽  
N Terminus ◽  
Μ Opioid Receptor ◽  
Magnesium Salts

<div>In the crystal structure of BU72 bound to the μ opioid receptor, the opioid clashes with an adjacent residue, and unexplained electron density connects the two. It has been reported that this density can be filled by a magnesium ion. However, this proposal requires 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: HEPES buffer, nickel ions, and an N-terminus that forms redox-active nickel complexes. Here I show that an oxygen atom fills the unexplained density, giving a known type of covalent adduct with reasonable geometry and no clashes. Strain is evident, but is consistent with tension from the tethered N-terminus.</div>

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

2021 ◽  
Author(s):  
Thomas A. Munro
Keyword(s):  
Crystal Structure ◽  
Opioid Receptor ◽  
Coordination Shell ◽  
Covalent Bonds ◽  
Nickel Ions ◽  
Redox Active ◽  
Covalent Adduct ◽  
N Terminus ◽  
Μ Opioid Receptor ◽  
Non Covalent Interactions

<div>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.</div>

scholarly journals Revised (β-phenyl) stereochemistry of ultrapotent μ opioid BU72

2020 ◽  
Author(s):  
Thomas A. Munro
Keyword(s):  
Crystal Structure ◽  
Electron Density ◽  
Opioid Receptor ◽  
Phenyl Group ◽  
Benzylic Carbon ◽  
Μ Opioid Receptor ◽  
Correct Structure

AbstractIn its crystal structure in complex with the μ opioid receptor (μOR), the opioid BU72 exhibits extreme deviations from the expected geometry.1 Three of these involve the phenyl group. There is also unexplained electron density next to the benzylic carbon. Here I show that inverting the benzylic configuration fills this unexplained density and eliminates the phenyl group outliers, along with all but one of the others. I propose that this is the correct structure of BU72.

scholarly journals μ-Opioid receptor-stimulated synthesis of reactive oxygen species is mediated via phospholipase D2

2009 ◽  
Vol 110 (4) ◽  
pp. 1288-1296 ◽  
Author(s):  
Thomas Koch ◽  
Anja Seifert ◽  
Dai-Fei Wu ◽  
Marija Rankovic ◽  
Jürgen Kraus ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Opioid Receptor ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Phospholipase D2 ◽  
Μ Opioid Receptor

scholarly journals Characterization of a Computationally Designed Water-soluble Human μ-Opioid Receptor Variant Using Available Structural Information

2014 ◽  
Vol 121 (4) ◽  
pp. 866-875 ◽  
Author(s):  
Xuelian Zhao ◽  
Jose Manuel Perez-Aguilar ◽  
Felipe Matsunaga ◽  
Mitchell Lerner ◽  
Jin Xi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Opioid Receptor ◽  
Structural Information ◽  
Water Soluble ◽  
High Yield ◽  
New Variant ◽  
Helical Content ◽  
Novel Variant ◽  
Μ Opioid Receptor ◽  
Increasing Temperature

Abstract Background: The recent X-ray crystal structure of the murine μ-opioid receptor (MUR) allowed the authors to reengineer a previously designed water-soluble variant of the transmembrane portion of the human MUR (wsMUR-TM). Methods: The new variant of water-soluble MUR (wsMUR-TM_v2) was engineered based on the murine MUR crystal structure. This novel variant was expressed in Escherichia coli and purified. The properties of the receptor were characterized and compared with those of wsMUR-TM. Results: Seven residues originally included for mutation in the design of the wsMUR-TM were reverted to their native identities. wsMUR-TM_v2 contains 16% mutations of the total sequence. It was overexpressed and purified with high yield. Although dimers and higher oligomers were observed to form over time, the wsMUR-TM_v2 stayed predominantly monomeric at concentrations as high as 7.5 mg/ml in buffer within a 2-month period. Its secondary structure was predominantly helical and comparable with those of both the original wsMUR-TM variant and the native MUR. The binding affinity of wsMUR-TM_v2 for naltrexone (Kd approximately 70 nM) was in close agreement with that for wsMUR-TM. The helical content of wsMUR-TM_v2 decreased cooperatively with increasing temperature, and the introduction of sucrose was able to stabilize the protein. Conclusions: A novel functional wsMUR-TM_v2 with only 16% mutations was successfully engineered, expressed in E. coli, and purified based on information from the crystal structure of murine MUR. This not only provides a novel alternative tool for MUR studies in solution conditions but also offers valuable information for protein engineering and structure–function relations.

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