New ligand-binding sites identified in the crystal structures of β-lactoglobulin complexes with desipramine

Abstract

The homodimeric β-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 β-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 β-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 FAW–DSM complexes together with data from the existing literature are used to establish a systematic classification of the ligand-binding sites in the β-lactoglobulin molecule.

β‐Lactoglobulin variants as potential carriers of pramoxine: Comprehensive structural and biophysical studies

“…The binding sites localized at the dimer interface was identified for the BLG‐vitamin D3 complex (occ = 0.5; PDB ID: 2GJ5) 71 and for structure of the engineered BLG (variant I56F/L39A/M107F) with desipramine (occ = 1.0; PDB ID: 7Q18). 27 The modification of the binding pocket shape via mutations proved to be a successful tool for creation of the cavity capable to accommodate large molecules as mentioned desipramine (occ = 1.0; PDB ID: 7Q19) 27 and chlorpromazine (occ = 0.75, PDB ID: 5NUJ). 21 Another interesting example of the modulation of the BLG‐binding mode is a set of variants, that can be classified according to their binding properties to tetracaine 26 .…”

“…PDB ID: 7Q18). 27 The modification of the binding pocket shape via mutations proved to be a successful tool for creation of the cavity to accommodate large molecules as mentioned desipramine (occ = 1.0; PDB ID: 7Q19) 27 and chlorpromazine (occ = 0.75, PDB ID: 5NUJ). 21 Another interesting example of the modulation of the BLG-binding mode is a set of variants, that can be classified according to their binding properties to tetracaine.…”

“…23,24 Several new BLG variants containing single or multiple mutations within the binding pocket have previously been developed. 21,[25][26][27] These proteins were modified to increase the selectivity of binding of bioactive compounds, in particular drugs possessing aromatic fragments. The type of amino acid substitution was selected via a structure-based approach using a three-dimensional alignment of proteins with almost identical folds but low sequence identity.…”

“…Several new BLG variants containing single or multiple mutations within the binding pocket have previously been developed 21,25–27 . These proteins were modified to increase the selectivity of binding of bioactive compounds, in particular drugs possessing aromatic fragments.…”

Estudo do acoplamento molecular β-lactoglobulina/vanilina – Abordagens espectroscópica e por simulações computacionais de docking

“…• O fármaco tricíclico desipramina (DSM) esteve presente nos três sítios de interação conhecidos nas estruturas diméricas de β-lg, sendo que no sítio no interior do β-barril possuiu a maior afinidade pelo DSM (LOCH et al, 2022);…”