Mechanism of signal transduction through the <italic>Escherichia coli</italic> two-component system transmitter protein NRII (NTRB).

Bacteria thrive in diverse environments. Their ability to adapt to drastically changing environmental conditions stems in part from their widespread use of Two-component signal transduction systems (TCS). A TCS consists of two proteins, the transmitter and the receiver, that use a conserved phosphoryl transfer mechanism to regulate a biological process in response to environmental signals. Environmental signals control the transmitter's ability to phosphorylate and dephosphorylate its cognate receiver, and the phosphorylation state of the receiver determines its ability to elicit the proper response. Our studies of the paradigmatic NRII/NRI TCS of Escherichia coli revealed the mechanism of signal transduction through the transmitter NRII. Intracellular signals of nitrogen and carbon status control the ability of the PII protein to regulate NRII. PII inhibits the kinase activity and activates the phosphatase activity of NRII. Each subunit of the homodimeric NRII contains three domains; an unconserved N-terminal domain, and a central and C-terminal domain that are conserved among the transmitter family. Cross-linking studies with purified components demonstrated that the binding of PII to NRII depended on small molecule effectors that control the ability of PII to regulate NRII. The purified cross-linked complex, which exhibited phosphatase activity, appeared to consist of a PII trimer linked to a single subunit of the NRII dimer. The isolated C-terminal domain of NRII was sufficient for PII-cross-linking. A genetic selection and screening procedure identified many single amino acid substitutions in NRII that resulted in diminished phosphatase activity, while having little effect on kinase activity. The mutations mapped throughout all three domains of NRII including two regions of the C-terminal domain, the ATP-lid and a surface on the back of this domain. A mutation in the latter surface resulted in severely diminished PII-binding. Using purified components, heterodimers were formed that contained subunits from different phosphatase-deficient NRII proteins. Certain combinations of mutations resulted in trans-intramolecular complementation partially restoring phosphatase activity. The pattern of complementation was consistent with a model where the phosphatase activity utilizes a PII-binding site from one subunit of the NRII dimer, a central domain from the same subunit, and a C-terminal domain ATP-lid from the opposing subunit.Ph.D.BiochemistryBiological SciencesMicrobiologyPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123765/2/3101149.pd