Journal
IUCRJ
Volume 9, Issue -, Pages 386-398Publisher
INT UNION CRYSTALLOGRAPHY
DOI: 10.1107/S2052252522004183
Keywords
beta-lactoglobulin; desipramine; ligand-binding sites
Funding
- NIGMS [R01-GM132595]
- NIH [HG008424]
- program `Excellence Initiative -Research University' at the Jagiellonian University in Krakow
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This study identified alternative ligand-binding sites in the beta-lactoglobulin molecule, providing important evidence through crystal structure analysis. The importance of shape complementarity for ligand recognition and selectivity was highlighted in the analysis.
The homodimeric beta-lactoglobulin belongs to the lipocalin family of proteins that transport a wide range of hydrophobic molecules and can be modified by mutagenesis to develop specificity for novel groups of ligands. In this work, new lactoglobulin variants, FAF (I56F/L39A/M107F) and FAW (I56F/L39A/M107W), were produced and their interactions with the tricyclic drug desipramine (DSM) were studied using X-ray crystallography, calorimetry (ITC) and circular dichroism (CD). The ITC and CD data showed micromolar affinity of the mutants for DSM and interactions according to the classical one-site binding model. However, the crystal structures unambiguously showed that the FAF and FAW dimers are capable of binding DSM not only inside the beta-barrel as expected, but also at the dimer interface and at the entrance to the binding pocket. The presented high-resolution crystal structures therefore provide important evidence of the existence of alternative ligand-binding sites in the beta-lactoglobulin molecule. Analysis of the crystal structures highlighted the importance of shape complementarity for ligand recognition and selectivity. The binding sites identified in the crystal structures of the FAF-DSM and FAWDSM complexes together with data from the existing literature are used to establish a systematic classification of the ligand-binding sites in the beta-lactoglobulin molecule.
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