NMR Studies On The Phosphorvlation Mechanism Of N-terminus Of Response Regulator YycF From Bacillus Subtilis

The YycGF system, originally identified in Bacillus subtilis, has been recognized as a crucial two-component signal transduction system closely associated with cell viability. It is highly conserved in low G+C gram-positive bacteria, including Staphylococcus aureus, Streptococcus pneumoniae and other human pathogens. The histidine protein kinase (HK) YycG senses extracellular or intracellular signals and phosphorylates its cognate response regulator (RR) YycF, which in turn recognizes sequence-specific regions on the bacterial chromosome and regulates the expression of certain genes. The presence of a divalent metal ion is essential for phosphoryl group transfer. Here, we presented NMR (Nuclear Magnetic Resonance) studies on the N-terminus of response regulator YycF from Bacillus subtilis. The results will provide important clues for understanding the signal transduction process of this protein.The N-terminal receiver domain of YycF (YycFN) from Bacillus subtilis was overexpressed and purified 1H-15N HSQC spectra showed that the addition of magnesium ions induced significant conformational changes of YycFN. By using heteronuclear multidimensional NMR methods, we collected and processed NMR data of 13C,15N-labelled YycFN, and obtained most of the backbone chemical shift assignments in the absence and presence of Mg2+.Four divalent metal ions, Ca2+, Mg2+, Cu2+ and Mn2+, were titrated into the solution of YycFN, respectively. The metal ions of Ca2+ and Mg2+ induced severe chemical shift changes of YycFN backbone resonances, which were mainly located at Asp9, Asp16 and the phosphorylated site of Asp53. The β34a4 loop was also perturbed by the metal binding. The dissociation constant (Kd) of Ca2+ and Mg2+ with YycFN were calculated by fitting the chemical shift changes against the metal ion concentration. The dynamics of YycFN was also investigated in the absence and presence of metal ions. For paramagnetic metal ions of Cu2+ and Mn2+, titrations were followed by carefully monitoring peak intensity changes and line broadening. Differential line broadening in the 1H-15N HSQC spectra, due to relaxation processes involving an unpaired electron from the metal ion, can be used to easily localize sites of paramagnetic ion binding.A typical response regulator accepts the phosphoryl group from the cognate histidine kinase and triggers conformational changes as a mechanism for activation. However, the activation mechanism is unclear for YycFN. We used YycG and ATP to catalyze the phosphorylation of YycFN. Chemical shift changes were observed for the residues close to the phosphorylation site, and the presence of a divalent metal ion is essential in the interaction between YycG and YycFN. Further studies need to be carried out to investigate the conformational change of YycFN induced by YycG binding and their phosphotransfer process.