PilZ domain as a c-di-GMP binding receptor

Abstract

Cyclic-di-GMP, a ubiquitous intracellular bacterial signaling molecule, is responsible for regulating biofilm formation, motility and multicellular behavior of diverse bacteria. This molecule is produced from two molecules of GTP by diguanylate cyclases (DGCs) of GGDEF domain and degraded to GMP via the linear molecule pGpG by phosphodiesterases (PDEs) of EAL domain. GGDEF and EAL domains are found in large numbers in most bacterial species and known to regulate c-di-GMP dependent signaling, however no protein has been shown to bind c-di-GMP and the targets for c-di-GMP action remain unknown. Amikam and Gaperin lately suggested PilZ domain is part of the long sought c-di-GMP binding protein. Here in this study, structural evidence that c-di-GMP indeed binds to the PilZ domain was provided. Several proteins containing PilZ domain were expressed and purified. Some were tested its HSQC while others were crystallized to find out whether they bind to c-di-GMP or not. PA4608 was expressed and purified beautifully and it was found to bind c-di-GMP with monomer state. PP4397, PilZN domain and PilZ domain of E.coli YcgR were over-expressed and purified to be checked their binding with c-di-GMP and its oligomeric states during the binding.