Ma Dejian, Tillman Tommy S, Tang Pei, Meirovitch Eva, Eckenhoff Roderic, Carnini Anna, Xu Yan
Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA.
Proc Natl Acad Sci U S A. 2008 Oct 28;105(43):16537-42. doi: 10.1073/pnas.0805501105. Epub 2008 Oct 23.
Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics.
多结构域膜蛋白的结构研究常常受到这些蛋白在膜模拟环境中的多变性以及用传统技术处理它们的困难的阻碍。设计并创建具有保留的天然结构的水溶性类似物提供了一种有吸引力的替代方法。我们在此报告对钾通道KcsA的水溶性类似物WSK3的溶液核磁共振研究。WSK3的核磁共振结构（蛋白质数据银行ID代码2K1E）与KcsA的晶体结构相似，验证了该方法。然而，通过更严格的比较标准，为提高水溶性而引入的几个带电荷残基似乎导致了一些天然结构中不存在的盐桥和氢键的可能形成，从而导致WSK3相对于KcsA的结构略有差异。核磁共振动力学测量表明，WSK3在没有脂质环境的情况下具有高度的灵活性。降低的光谱密度映射和无模型分析揭示了与经历缓慢（纳秒级）运动且局部有序性异常低的各向同性翻滚四聚体一致的动态特征。选择性过滤器和孔螺旋附近的氢键网络改变，以及选择性过滤器的内在动态性质，支持了该区域对缓慢失活至关重要的观点。我们的结果不仅对膜蛋白水溶性类似物的设计有影响，而且对我们理解蛋白质内在结构和动力学的基本决定因素也有影响。
