Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
FEBS J. 2014 May;281(10):2422-30. doi: 10.1111/febs.12795. Epub 2014 Apr 17.
In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because L-Glu is difficult to displace, despite extensive dialysis. Here, we show that L-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mM) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mM), and we use differential scanning fluorimetry to show that L-Asp is able to stabilize the isolated GluA2 LBD. We also show that L-Asp can replace L-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mM L-Asp. Surprisingly, with 7.5 mM L-Asp, the GluA2 LBD crystallized as a mixed dimer, with L-Glu being present in one subunit, and neither L-Asp nor L-Glu being present in the other subunit. Thus, residual L-Glu is retained from the expression medium. On the other hand, only L-Asp was found at the binding site when 50 or 250 mM L-Asp was used for crystallization. The binding mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taking our findings together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design.
Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mM L-Asp), 4O3C (50 mM L-Asp), and 4O3A (250 mM L-Asp).
在离子型谷氨酸受体 A2 (GluA2) 配体结合域 (LBD) 的纯化过程中，先前曾使用 L-Glu 补充缓冲液来稳定蛋白质。这有时会阻碍低亲和力配体的结构研究，因为尽管进行了广泛的透析，L-Glu 仍难以置换。在这里，我们表明 L-Asp 与全长 GluA2 的结合亲和力较低（Ki = 0.63 mM），与 GluA2 LBD 的结合亲和力更低（Ki = 2.6 mM），并且我们使用差示扫描荧光法表明 L-Asp 能够稳定分离的 GluA2 LBD。我们还表明，L-Asp 可以在纯化过程中替代 L-Glu，从而提供相同的产量和所得蛋白质样品的纯度。此外，我们在存在 7.5、50 和 250 mM L-Asp 的情况下解决了 GluA2 LBD 的三个结构。令人惊讶的是，在存在 7.5 mM L-Asp 的情况下，GluA2 LBD 结晶为混合二聚体，一个亚基中存在 L-Glu，另一个亚基中既不存在 L-Asp 也不存在 L-Glu。因此，表达培养基中仍保留残留的 L-Glu。另一方面，当使用 50 或 250 mM L-Asp 进行结晶时，仅在结合位点发现 L-Asp。在 GluA2 LBD 上观察到的 L-Asp 结合模式与 L-Glu 描述的模式非常相似。综合我们的研究结果，我们已经表明，L-Asp 可以在纯化过程中替代 L-Glu，用于依赖配体的 GluA2 LBD 稳定化。这将能够进行低亲和力配体的结构研究，以优化基于结构的药物设计中的先导化合物。
结构数据可在蛋白质数据库 (PDB) 中以 4O3B（7.5 mM L-Asp）、4O3C（50 mM L-Asp）和 4O3A（250 mM L-Asp）的形式获得。
