Visualizing the Pre-Active Conformation of Response Regulator PhoBNF20D in Its apo State

Abstract

PhoB is a response regulator of the PhoR/PhoB two-component system (TCS) in Escherichia Coli, which is involved in environmental phosphate regulation. PhoB activation is known to be mediated by Mg2+ coordination and phosphorylation of the conserved D53 residue, but the exact mechanism remains to be elucidated. It has been proposed that minor active conformations of the response regulator may preexist in its apo state and be related to the activation. However, such minor states are instable and difficult to observe. In this work, 1H-15N HSQC and 19F NMR experiments were performed on a monomeric mutant PhoBNF20D. It was observed that the apo PhoBNF20D had a pre-active conformation, which could be partially stabilized by the phosphoryl group analog BeF3-without the presence of Mg2+. Addition of Mg2+ shifted the equilibrium towards the active form. Furthermore, the exchange between the inactive and active conformations were confirmed by the CPMG (Carr-Purcell-Meiboom-Gill) relaxation dispersion experiments. It is concluded that the apo PhoBNF20D have both inactive and active conformations, and the pre-active conformation can be stabilized by BeF3-.

Keywords

• BeF3-
• active conformation
• nuclear magnetic resonance (NMR)
• response regulator
• two-component system (TCS)