In vitro phosphotransfer in the NmpRSTU multi-component pathway.

(A) The kinases NmpS and NmpU autophosphorylate in the presence of ATP-γP³² as seen by the accumulation of the radioactive phosphoryl group. NmpU specifically phosphorylates the first receiver domain of NmpT (RR1) and the receiver domain of NmpS (B and C). In contrast, NmpS only phosphorylates NmpR (D and E). Note the rapid kinetics of the phosphorylation of NmpR by NmpS (E). Also, the V87E amino acid change of NmpR does not affect the specificity of the phosphotransfer (C and E). The specificity residues [10, 11, 96] of the SK domains of NmpS and NmpU and the corresponding specificity residues of the receiver domains of NmpR, NmpS, and NmpT further supports the observed phosphotransfer patterns (F).