Citrate Sensing by the C4-Dicarboxylate/Citrate Sensor Kinase DcuS of Escherichia coli: Binding Site and Conversion of DcuS to a C4-Dicarboxylate- or Citrate-Specific Sensor

摘要

The histidine protein kinase DcuS of Escherichia coli senses C4-dicarboxylates and citrate by a periplasmic domain. The closely related sensor kinase CitA binds citrate, but no C4-dicarboxylates, by a homologous periplasmic domain. CitA is known to bind the three carboxylate and the hydroxyl groups of citrate by sites C1, C2, C3, and H. DcuS requires the same sites for C4-dicarboxylate sensing, but only C2 and C3 are highly conserved. It is shown here that sensing of citrate by DcuS required the same sites. Binding of citrate to DcuS, therefore, was similar to binding of C4-dicarboxylates but different from that of citrate binding in CitA. DcuS could be converted to a C4-dicarboxylate-specific sensor (DcuSDC) by mutating residues of sites C1 and C3 or of some DcuS-subtype specific residues. Mutations around site C1 aimed at increasing the size and accessibility of the site converted DcuS to a citrate-specific sensor (DcuSCit). DcuSDC and DcuSCit had complementary effector specificities and responded either to C4-dicarboxylates or to citrate and mesaconate. The results imply that DcuS binds citrate (similar to the C4-dicarboxylates) via the C4-dicarboxylate part of the molecule. Sites C2 and C3 are essential for binding of two carboxylic groups of citrate or of C4-dicarboxylates; sites C1 and H are required for other essential purposes.