A Predicted Structure for the PixD鈥揚ixE Complex Determined by Homology Modeling, Docking Simulations, and a Mutagenesis Study

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• 作者：Shukun Ren ; Ryoichi Sato ; Koji Hasegawa ; Hiroyuki Ohta ; Shinji Masuda
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：February 19, 2013
• 年：2013
• 卷：52
• 期：7
• 页码：1272-1279
• 全文大小：417K
• 年卷期：v.52,no.7(February 19, 2013)
• ISSN：1520-4995

文摘

PixD is a blue light-using flavin (BLUF) photoreceptor that controls phototaxis in the cyanobacterium Synechocystis sp. PCC6803. PixD interacts with the response regulator-like protein PixE in a light-dependent manner, and this interaction is critical for light signal transduction in vivo. However, the structure of the PixD鈥揚ixE complex has not been determined. To improve our understanding of how PixD transmits its captured light signal to PixE, we used blue-native polyacrylamide gel electrophoresis to characterize the molecular mass of a recombinant PixD鈥揚ixE complex purified from Escherichia coli and found it to be 342 kDa, suggesting that the complex contains 10 PixD and 4 PixE monomers. The stoichiometry of the complex was confirmed by Western blotting. Specifically, three intermediate states, PixD₁₀鈥揚ixE₁, PixD₁₀鈥揚ixE₂, and PixD₁₀鈥揚ixE₃, were detected. The apparent dissociation constant for PixE and PixD is 5 渭M. A docking simulation was performed using a modeled PixE structure and the PixD₁₀ crystal structure. The docking simulation showed how the molecules in the PixD₁₀鈥揚ixE₄ structure interact. To verify the accuracy of the docked model, a site-directed mutagenesis study was performed in which Arg80 of PixE, which appears to be capable of interacting electrostatically with Asp135 of PixD in the predicted structure, was shown to be critical for complex formation as mutation of PixE Arg80 to Asp or Ala prevented PixD鈥揚ixE complex formation. This study provides a structural basis for future investigations of the light signal transduction mechanism involving PixD and PixE.