Durell S R, Hao Y, Guy H R
Laboratory of Experimental and Computational Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-5567, USA.
J Struct Biol. 1998;121(2):263-84. doi: 10.1006/jsbi.1998.3962.
A large collaborative, multidisciplinary effort involving many research laboratories continues which uses indirect methods of molecular biology and membrane biophysics to analyze the three-dimensional structures and functional mechanisms of K+ channels. This work also extends to the distant relatives of these channels, including the voltage-gated Na+ and Ca2+ channels. The role that our group plays in this process is to combine the information gained from experimental studies with molecular modeling techniques to generate atomic-scale structural models of these proteins. The modeling process involves three stages which are summarized as: (I) prediction of the channel sequence transmembrane topology, including the functionality and secondary structure of the segments; (II) prediction of the relative positions of the transmembrane segments, and (III) filling in all atoms of the amino acid residues, with conformations for energetically stabilized interactions. Both physiochemical and evolutionary principles (including sequence homology analysis) are used to guide the development. In addition to testing the steric and energetic feasibilities of different structural hypotheses, the models provide guidance for the design of new experiments. Structural modeling also serves to "fill in the gaps" of experimental data, such as predicting additional residue interactions and conformational changes responsible for functional processes. The modeling process is currently at the stage that experimental studies have definitely confirmed most of our earlier predictions about the transmembrane topology and functionality of different segments. Additionally, this report describes the detailed, three-dimensional models we have developed for the entire transmembrane region and important functional sites of the voltage-gated Shaker K+ channel in the open, closed, and inactivated conformations (including the ion-selective pore and voltage-sensor regions). As part of this effort, we also describe how our development of structural models for many of the other major K+ channel families aids in determining common structural motifs. As an example, we also present a detailed model of the smaller, bacterial K+ channel from Streptomyces lividans. Finally, we discuss strategies for using newly developed experimental methods for determining the structures and analyzing the functions of these channel proteins.
一项涉及众多研究实验室的大规模跨学科合作仍在继续，该合作运用分子生物学和膜生物物理学的间接方法来分析钾离子通道的三维结构和功能机制。这项工作还拓展到了这些通道的远亲，包括电压门控钠离子通道和钙离子通道。我们团队在这个过程中所起的作用是将实验研究获得的信息与分子建模技术相结合，以生成这些蛋白质的原子尺度结构模型。建模过程包括三个阶段，总结如下：（I）预测通道序列的跨膜拓扑结构，包括各片段的功能和二级结构；（II）预测跨膜片段的相对位置；（III）填充氨基酸残基的所有原子，并确定能实现能量稳定相互作用的构象。物理化学原理和进化原理（包括序列同源性分析）都被用于指导模型的构建。除了测试不同结构假设的空间和能量可行性外，这些模型还为新实验的设计提供指导。结构建模还用于“填补”实验数据的空白，比如预测导致功能过程的额外残基相互作用和构象变化。目前建模过程处于这样一个阶段：实验研究已明确证实了我们之前对不同片段跨膜拓扑结构和功能的大部分预测。此外，本报告描述了我们针对电压门控Shaker钾离子通道在开放、关闭和失活构象下（包括离子选择性孔道和电压感受器区域）的整个跨膜区域及重要功能位点所构建的详细三维模型。作为这项工作的一部分，我们还描述了我们为许多其他主要钾离子通道家族构建结构模型如何有助于确定共同的结构基序。例如，我们还展示了来自淡紫链霉菌的较小细菌钾离子通道的详细模型。最后，我们讨论了使用新开发的实验方法来确定这些通道蛋白结构并分析其功能的策略。
