Loch Joanna I, Barciszewski Jakub, Śliwiak Joanna, Bonarek Piotr, Wróbel Paulina, Pokrywka Kinga, Shabalin Ivan G, Minor Wladek, Jaskolski Mariusz, Lewiński Krzysztof
Department of Crystal Chemistry and Crystal Physics, Faculty of Chemistry, Jagiellonian University, Kraków, Poland.
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.
IUCrJ. 2022 Apr 29;9(Pt 3):386-398. doi: 10.1107/S2052252522004183. eCollection 2022 May 1.
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.
同二聚体β-乳球蛋白属于脂质运载蛋白家族，该家族蛋白可转运多种疏水分子，并且可以通过诱变进行修饰，以产生对新型配体组的特异性。在这项工作中，制备了新的乳球蛋白变体FAF（I56F/L39A/M107F）和FAW（I56F/L39A/M107W），并使用X射线晶体学、量热法（ITC）和圆二色性（CD）研究了它们与三环药物地昔帕明（DSM）的相互作用。ITC和CD数据显示突变体对DSM具有微摩尔亲和力，并根据经典的单点结合模型发生相互作用。然而，晶体结构明确表明，FAF和FAW二聚体不仅能够如预期那样在β-桶内部结合DSM，还能在二聚体界面和结合口袋入口处结合DSM。因此，所呈现的高分辨率晶体结构为β-乳球蛋白分子中存在替代配体结合位点提供了重要证据。对晶体结构的分析突出了形状互补性对配体识别和选择性的重要性。FAF-DSM和FAW-DSM复合物晶体结构中确定的结合位点以及现有文献的数据被用于建立β-乳球蛋白分子中配体结合位点的系统分类。
