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A predicted structure for the PixD-PixE complex determined by homology modeling, docking simulations, and a mutagenesis study.
MedLine Citation:
PMID:  23346988     Owner:  NLM     Status:  Publisher    
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-PixE complex has not been solved. To better understand 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-PixE 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-PixE1, PixD10-PixE2, and PixD10-PixE3, were detected. The apparent dissociation constant for PixE and PixD is ~5 μM. A docking simulation was performed using a modeled PixE structure and the PixD10 crystal structure. The docking simulation showed how the molecules in PixD10/PixE4 interact. To verify the accuracy of the docked model, a site-directed mutagenesis study was performed in which Arg80 of PixE, which appears capable of interacting electrostatically with Asp135 of PixD in the predicted structure, was shown to be critical for the complex formation as mutation of PixE Arg80 to Asp or Ala prevented PixD-PixE complex formation. This study provides a structural basis for future investigations of the light-signal transduction mechanism involving PixD and PixE.
Shukun Ren; Ryoichi Sato; Koji Hasegawa; Hiroyuki Ohta; Shinji Masuda
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-25
Journal Detail:
Title:  Biochemistry     Volume:  -     ISSN:  1520-4995     ISO Abbreviation:  Biochemistry     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-25     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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