STRUCTURAL AND FUNCTIONAL ANALYSES OF ACTIVATING AMINO-ACID SUBSTITUTIONS IN THE RECEIVER DOMAIN OF NTRC - EVIDENCE FOR AN ACTIVATING SURFACE

The bacterial enhancer-binding protein NtrC activates transcription wh en phosphorylated on aspartate 54 in its amino (N)-terminal regulatory domain or when altered by constitutively activating amino acid substi tutions. The N-terminal domain of NtrC, which acts positively on the r emainder of the protein, is homologous to a large family of signal tra nsduction domains called receiver domains. Phosphorylation of an aspar tate residue in a receiver domain modulates the function of downstream target, but the accompanying structural changes are not clear. In the present work we examine structural and functional differences between the wild-type receiver domain of NtrC and mutant forms carrying const itutively activating substitutions. Combinations of such substitutions resulted in both increased structural changes in the N-terminal domai n, monitored by NMR chemical shift differences, and increased transcri ptional activation by the full-length protein. Structural changes caus ed by substitutions outside the active site (D86N and A89T) were not o nly local but extended over a substantial portion of the N-terminal do main including the region from alpha-helix 3 to beta-strand 5 (''3445 face'') and propagating to the active site. Interestingly, the activat ing substitution of glutamate for aspartate at the site of phosphoryla tion (D54E) also triggered structural changes in the 3445 face. Thus, the active site and the 3445 face appear to interact. Implications wit h respect to how phosphorylation may affect the structure of receiver domains and how structural changes may be communicated to the remainde r of NtrC are discussed. (C) 1997 Academic Press Limited.