Cross‐linking analysis reveals the putative dimer structure of the cyanobacterial BLUF photoreceptor PixD

Abstract

a b s t r a c tPixD is a blue light using flavin (BLUF)-type blue-light photoreceptor controlling phototaxis in the cyanobacterium Synechocystis sp. PCC6803. The crystal structure of PixD shows a decamer, although in solution an equilibrium is maintained between the dimer and decamer. Because the ratio of these two conformers is altered by illumination, the equilibrium state determines photosensitivity. However, no structural information is available for the PixD dimer. Here, we report a predicted structure for the dimer based on docking simulation, mutagenesis, and mass spectrometry-based cross-linking analyses. The results indicate the importance of the PixD C-terminus for dimer preference and photosensitivity.

Light-induced chromophore and protein responses and mechanical signal transduction of BLUF proteins

“…These results underscore the importance of interactions between the C-terminus of two PixD molecules to form a dimer. When the C-terminal region of PixD is removed, PixD does not form a stable dimer, a finding that further supports the PixD dimeric structural model (Ren et al 2015 ). The in-silico modeled PixD dimer is not similar to any dimeric combination found in the PixD crystal structure (Fig.…”

“…Crystalline PixD forms a decamer in which two pentameric rings are stacked face-to-face (Yuan et al 2006 ). Gel-filtration and blue-native PAGE studies have revealed that purified PixD equilibrates between a dimer and decamer in solution (Ren et al 2013 , 2015 ; Tanaka et al 2011 ; Yuan and Bauer 2008 ), suggesting that the crystal structure of decameric PixD possibly reflects a decameric structure in vivo. Conversely, a solution structure for dimeric PixD has not been established.…”

“…The flavin chromophores are indicated as ball-and-stick structures. Reproduced from (Ren et al 2015 ) with a slight modification …”

“…Ren and co-workers cross-linked purified PixD and examined the products by mass spectrometry (Ren et al 2015 ). They found that two residues, Glu26 and Lys22, were cross-linked with a zero-length cross-linker to form a covalent dimer, suggesting that these two residues from different molecules are positioned closely in the PixD dimer.…”

“…They found that two residues, Glu26 and Lys22, were cross-linked with a zero-length cross-linker to form a covalent dimer, suggesting that these two residues from different molecules are positioned closely in the PixD dimer. They also performed docking simulations to characterize possible intermolecular interactions in a dimeric PixD structure; their results indicated that Glu26 and Lys22 could be closely positioned and that the C-terminal regions of two PixD molecules can interact (Ren et al 2015 ). These results underscore the importance of interactions between the C-terminus of two PixD molecules to form a dimer.…”