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Updated: Jan 21, 2026
Identification of Functional Protein Regions Through Chimeric Protein Construction
Published on: January 8, 2019
Characterisation of the ligand binding sites in the translocator protein TSPO using the chimeric bacterial-mammalian
Elisabeth Graeber1, Volodymyr M Korkhov2
1Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland.
Abstract:
The translocator protein TSPO is in an important diagnostic and therapeutic target in a range of pathologies, including neuroinflammation and cancer. Despite the availability of several structures of TSPO homologues, our understanding of the molecular determinants that govern high-affinity interactions of TSPO with its ligands is incomplete. Here, in order to decipher the key structural elements of TSPO responsible for interactions with its ligands, we designed a panel of chimeric proteins mimicking the mammalian substrate binding site grafted onto the backbone of the Rhodobacter sphaeroides TSPO homologue, RsTSPO. One of the designed chimeric constructs, RsMouse, could be heterologously expressed and displayed improved binding affinities for the known TSPO drugs diazepam, PK11195 and NBD-FGIN-1-27. Furthermore, the chimeric protein had improved interactions with NBD-cholesterol, a fluorescent analogue of the presumed natural substrate of TSPO. Partial modifications of the transmembrane helix bundle in the chimeric construct differentially affected binding of the TSPO drugs and the natural substrates of TSPO, consistent with the presence of multiple ligand binding sites in the protein. Based on the available structures of TSPO homologues, the substrate interactions may involve a lateral opening of the protein in the TM1-3, and stabilisation of TM4-5 is important for drug-like ligand binding. These observations are consistent with our experimental results, which show that the determinants of high-affinity ligand interactions of TSPO are distinct for different classes of ligands.
Insights
Researchers engineered chimeric proteins to understand how the translocator protein (TSPO) binds to ligands. This revealed distinct structural requirements for TSPO
Area of Science:
- Structural Biology
- Biochemistry
- Neuroinflammation and Cancer Research
Background:
- The translocator protein (TSPO) is a crucial target for diagnosing and treating neuroinflammation and cancer.
- Existing TSPO structures offer limited insight into the molecular basis of high-affinity ligand interactions.
Purpose of the Study:
- To elucidate the key structural elements of TSPO responsible for ligand binding.
- To investigate the differential binding affinities of various ligands to engineered TSPO variants.
Main Methods:
- Designed chimeric proteins by grafting mammalian TSPO binding sites onto the Rhodobacter sphaeroides TSPO (RsTSPO) backbone.
- Heterologous expression and characterization of chimeric constructs, including binding affinity assays for known drugs and cholesterol analogues.
- Analysis of structural modifications within the transmembrane helix bundle to assess their impact on ligand binding.
Main Results:
- The engineered RsMouse chimeric protein exhibited enhanced binding affinities for TSPO drugs (diazepam, PK11195, NBD-FGIN-1-27) and NBD-cholesterol.
- Modifications in the transmembrane helix bundle differentially affected the binding of drugs versus natural substrates, suggesting multiple binding sites.
- Structural insights suggest ligand interactions involve a lateral opening in TM1-3, with TM4-5 stabilization crucial for drug-like ligand binding.
Conclusions:
- The determinants governing high-affinity ligand interactions in TSPO are distinct for different ligand classes.
- Engineered chimeric proteins serve as valuable tools for dissecting TSPO structure-function relationships.
- Findings provide a foundation for developing more selective TSPO-targeting therapeutics.
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