Role of the H domain of the histidine kinase-like protein NifL in signal transmission.

The NifL protein from Azotobacter vinelandii senses both the redox and fixed nitrogen status to regulate nitrogen fixation by controlling the activity of the transcriptional activator NifA. NifL has a domain architecture similar to that of the cytoplasmic histidine protein kinases. It contains two N-terminal PAS domains and a C-terminal transmitter region containing a conserved histidine residue (H domain) and a nucleotide binding GHKL domain corresponding to the catalytic core of the histidine kinases. Despite these similarities, NifL does not exhibit kinase activity and regulates its partner NifA by direct protein-protein interactions rather than phosphorylation. NifL senses the redox status via a FAD co-factor located within the PAS1 domain and responds to the nitrogen status by interaction with the signal transduction protein GlnK, which binds to the GHKL domain. The ability of NifL to inhibit NifA is antagonized by the binding of 2-oxoglutarate to the N-terminal GAF domain of NifA. In this study we have performed site-directed mutagenesis of the H domain of NifL to examine its role in signal transmission. Our results suggest that this domain plays a major role in transmission of signals perceived by the PAS1 and GHKL domains to ensure that NifL achieves the required conformation necessary to inhibit the 2-oxoglutarate-bound form of NifA. Some of the substitutions discriminate the redox and fixed nitrogen sensing functions of NifL implying that the conformational requirements and/or domain interactions necessary for NifA inhibition differ with respect to the signal input.