Structural understanding of NtcA regulation and of its coactivation by the adaptor PII/NtcA shuttling protein PipX, which connects PII regulation with gene expression regulation

The CRP-FNR superfamily of transcriptional regulators includes the cyanobacterial master regulator NtcA, which orchestrates large responses of cyanobacteria to nitrogen scarcity. NtcA uses as allosteric activator 2-oxoglutarate (2OG), a signal of nitrogen poorness and carbon richness, and binds a coactivating protein (PipX) that shuttles between the signaling protein PII and NtcA depending on nitrogen richness, thus connecting PII signaling and gene expression regulation. Here, combining structural (X-ray crystallography of six types of crystals including NtcA complexes with DNA and 2OG and PipX), modelling and functional (EMSA and bacterial two-hybrid) studies, we clarify the binding and exquisite specificity of NtcA for its target DNA, its mechanisms of activation by 2OG, and its coactivation by PipX. Our crystal structures of PipX-NtcA-DNA complexes prove that PipX does not interact with DNA, although it increases NtcA-DNA contacts, and that it stabilizes the active, DNA-binding-competent conformation of NtcA. Superimposition of this complex on a very recently reported cryoEM structure of NtcA in a Transcription Activity Complex with RNA polymerase (RNAP), shows that PipX binding helps recruit RNAP by PipX interaction with RNAP, particularly with its gamma and sigma (region 4) subunits, a structural prediction supported here by bacterial two-hybrid experiments.