Lanigan Mark D, Kalman Katalin, Lefievre Yann, Pennington Michael W, Chandy K George, Norton Raymond S
Biomolecular Research Institute, Parkville 3052, Victoria, Australia.
Biochemistry. 2002 Oct 8;41(40):11963-71. doi: 10.1021/bi026400b.
The voltage-gated potassium channel in T lymphocytes, Kv1.3, an important target for immunosuppressants, is blocked by picomolar concentrations of the polypeptide ShK toxin and its analogue ShK-Dap22. ShK-Dap22 shows increased selectivity for Kv1.3, and our goal was to determine the molecular basis for this selectivity by probing the interactions of ShK and ShK-Dap22 with the pore and vestibule of Kv1.3. The free energies of interactions between toxin and channel residues were measured using mutant cycle analyses. These data, interpreted as approximate distance restraints, guided molecular dynamics simulations in which the toxins were docked with a model of Kv1.3 based on the crystal structure of the bacterial K(+)-channel KcsA. Despite the similar tertiary structures of the two ligands, the mutant cycle data imply that they make different contacts with Kv1.3, and they can be docked with the channel in configurations that are consistent with the mutant cycle data for each toxin but quite distinct from one another. ShK binds to Kv1.3 with Lys22 occupying the negatively charged pore of the channel, whereas the equivalent residue in ShK-Dap22 interacts with residues further out in the vestibule, producing a significant change in toxin orientation. The increased selectivity of ShK-Dap22 is achieved by strong interactions of Dap22 with His404 and Asp386 on Kv1.3, with only weak interactions between the channel pore and the toxin. Potent and specific blockade of Kv1.3 apparently occurs without insertion of a positively charged residue into the channel pore. Moreover, the finding that a single residue substitution alters the binding configuration emphasizes the need to obtain consistent data from multiple mutant cycle experiments in attempts to define protein interaction surfaces using these data.
T淋巴细胞中的电压门控钾通道Kv1.3是免疫抑制剂的重要靶点，皮摩尔浓度的多肽ShK毒素及其类似物ShK-Dap22可阻断该通道。ShK-Dap22对Kv1.3的选择性增强，我们的目标是通过探究ShK和ShK-Dap22与Kv1.3的孔道和前庭的相互作用，确定这种选择性的分子基础。利用突变循环分析测量毒素与通道残基之间相互作用的自由能。这些数据被解释为近似的距离限制，指导分子动力学模拟，其中毒素与基于细菌K(+)通道KcsA晶体结构的Kv1.3模型对接。尽管两种配体的三级结构相似，但突变循环数据表明它们与Kv1.3的接触不同，并且它们可以与通道以与每种毒素的突变循环数据一致但彼此截然不同的构型对接。ShK与Kv1.3结合时，Lys22占据通道带负电的孔道，而ShK-Dap22中的等效残基与前庭中更远的残基相互作用，导致毒素方向发生显著变化。ShK-Dap22选择性的增强是通过Dap22与Kv1.3上的His404和Asp386的强相互作用实现的，而通道孔道与毒素之间只有弱相互作用。Kv1.3的有效和特异性阻断显然是在没有带正电的残基插入通道孔道的情况下发生的。此外，单个残基取代改变结合构型的发现强调了在试图利用这些数据定义蛋白质相互作用表面时，需要从多个突变循环实验中获得一致的数据。
