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

Abstract

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, 2OG and PipX), modelling and functional (EMSA and bacterial two-hybrid) studies, we clarify the reasons for the exquisite specificity for the binding of NtcA to 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.