Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems.

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1. 24x10(6) M(-1).

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