Wang Wenning, Lo Kevin W-H, Kan Ho-Man, Fan Jing-Song, Zhang Mingjie
Department of Biochemistry, Molecular Neuroscience Center, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
J Biol Chem. 2003 Oct 17;278(42):41491-9. doi: 10.1074/jbc.M307118200. Epub 2003 Aug 6.
The 8-kDa light chain of dynein (DLC8) is ubiquitously expressed in various cell types. Other than serving as a light chain of the dynein complexes, this highly conserved protein has been shown to bind a larger number of proteins with diverse biological functions. DLC8 forms a homodimer via three-dimensional domain swapping of an internal beta-strand (the beta2-strand) at neutral pH. The protein undergoes non-reversible dimer-to-monomer dissociation when the pH value of the protein solution decreases. The three-dimensional structure of the DLC8 monomer determined by NMR spectroscopy at pH 3.0 showed that the protein is well folded. The major conformational change accompanied by dimer dissociation is in the beta2-strand of the protein, which undergoes transition from a beta-strand to a nascent alpha-helix. The monomer form of DLC8 is not capable of binding to target proteins. Insertion of two flexible amino acid residues in the tight beta1/beta2-loop dramatically stabilized the monomer conformation of the protein. NMR studies showed that the mutation altered the conformation as well as the three-dimensional domain swapping-mediated assembly of the DLC8 dimer. The mutant DLC8 was unable to bind to its targets even at physiological pH. The three-dimensional structure of the mutant protein in its monomeric form provides the structural basis of the mutation-induced stabilization of the monomer conformation. Based on the experimental data, we conclude that the formation of the beta2-strand swapping-mediated dimer is mandatory for the structure and function of DLC8. We further note that the DLC8 dimer represents a novel mode of three-dimensional domain swapping.
动力蛋白8 kDa轻链（DLC8）在多种细胞类型中普遍表达。除了作为动力蛋白复合物的轻链外，这种高度保守的蛋白质已被证明能与大量具有不同生物学功能的蛋白质结合。在中性pH条件下，DLC8通过内部β链（β2链）的三维结构域交换形成同型二聚体。当蛋白质溶液的pH值降低时，该蛋白质会发生不可逆的二聚体到单体的解离。在pH 3.0条件下通过核磁共振光谱法测定的DLC8单体的三维结构表明该蛋白质折叠良好。伴随二聚体解离的主要构象变化发生在蛋白质的β2链上，该链从β链转变为新生的α螺旋。DLC8的单体形式不能与靶蛋白结合。在紧密的β1/β2环中插入两个柔性氨基酸残基极大地稳定了蛋白质的单体构象。核磁共振研究表明，该突变改变了DLC8二聚体的构象以及三维结构域交换介导的组装。即使在生理pH条件下，突变型DLC8也无法与其靶标结合。突变蛋白单体形式的三维结构为突变诱导的单体构象稳定提供了结构基础。基于实验数据，我们得出结论，β2链交换介导的二聚体形成对于DLC8的结构和功能是必不可少的。我们进一步注意到，DLC8二聚体代表了一种新型的三维结构域交换模式。
