High mobility of carboxyl-terminal region of bacterial chemotaxis phosphatase CheZ is diminished upon binding divalent cation or CheY-P substrate.

In Escherichia coli chemotaxis, the CheZ phosphatase catalyzes the removal of the phosphoryl group from the signaling molecule, CheY. The cocrystal structure of CheZ with CheY x BeF3- x Mg2+ (a stable analogue of CheY-P) revealed that CheZ is a homodimer with a multidomain, nonglobular structure. To explore the effects of CheZ/CheY complex formation on CheZ structure, the rotational dynamics of the different structural domains of CheZ [the four-helix bundle, the N-terminal helix, the C-terminal helix, and the putative disordered linker between the C-terminal helix and the bundle] were evaluated. To monitor dynamics of the different regions, fluorescein probes were covalently attached at various locations on CheZ through reaction with engineered cysteine residues and the rotational behavior of the fluoresceinated derivatives were assessed using steady state fluorescence anisotropy. Anisotropy measurements at various solution viscosities (Perrin plot analysis) demonstrated large differences in global rotational motion for fluorophores located on different regions. Rotational correlation times for probes located on the four-helix bundle and the N-terminal helix agreed well with theoretical values predicted for a protein the size and shape of the four-helix bundle. However, the rotational correlation times of probes located on the linker and the C-terminal helix were 8-20x lower, indicating rapid motion independent of the bundle. The anisotropies of probes located on the linker and the C-terminal helix increased in the presence of divalent cation (Mg2+, Ca2+, or Mn2+) in a saturable fashion, consistent with a binding event (Kd approximately 1-4 mM) that results in decreased mobility. The anisotropies of probes located on the C-terminal helix and the C-terminal portion of the linker increased further as a result of binding CheY-P. In light of the recently available structural data and the high independent mobility of the C-terminus demonstrated here, we interpret the CheY-P-dependent increase in anisotropy to be a consequence of decreased mobility of the C-terminal region due to binding interactions with CheY-P, and not to the formation of higher order aggregates of the CheZ2(CheY-P)2 complex.