Two residues in the T-loop of GlnK determine NifL-dependent nitrogen control of nif gene expression

X-ray crystallographic analysis of the Escherichia coli P-II protein paralo gues GInB and GlnK has shown that they share a superimposable structural co re but can differ in conformation of the T-loop, a region of the protein (r esidues 37-54) that has been shown to be important for interaction with oth er proteins. In Klebsiella pneumoniae GlnK has been shown to have a clearly defined function in regulating NifL-mediated inhibition of NifA activity i n response to the nitrogen status, and GlnB, when expressed from the chromo some, does not substitute for GlnB Because the T-loops of K. pneumoniae and E. coli GlnB and GlnK differ at just three residues, 43, 52, and 54, we ha ve used a previously constructed heterologous system, in which a pneumoniae nifLA. is expressed in E. coli, to investigate the importance of GlnK resi dues 43, 52, and 54 for regulation of the NifLA interaction. By site-direct ed mutagenesis of glnB we have shown that residue 54 is the single most imp ortant amino acid in the T-loop in the context of the regulation of NifA ac tivity. Furthermore, a combination of just two changes, in residues 54 and 43, allows GlnB to function as GlnK and completely relieve NifL inhibition of NifA activity.