A predicted structure for the PixD-PixE complex determined by homology modeling, docking simulations, and a mutagenesis study.
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 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-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, PixD(10)-PixE(1), PixD(10)-PixE(2), and PixD(10)-PixE(3), 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 PixD(10) crystal structure. The docking simulation showed how the molecules in the PixD(10)-PixE(4) 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-PixE complex formation. This study provides a structural basis for future investigations of the light signal transduction mechanism involving PixD and PixE.
Center for Biological Resources and Informatics, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Shukun
S
Ren
Ryoichi
R
Sato
Koji
K
Hasegawa
Hiroyuki
H
Ohta
Shinji
S
Masuda
D000409:Alanine / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D055537:Light Signal Transduction; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D063347:Native Polyacrylamide Gel Electrophoresis; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein; D046939:Synechocystis / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D020283:Photoreceptors, Microbial; D011994:Recombinant Proteins; D001120:Arginine; D000409:Alanine