Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate

Abstract

2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena : the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.

SPR analysis of promoter binding of Synechocystis PCC6803 transcription factors NtcA and CRP suggests cross‐talk and sheds light on regulation by effector molecules

“…4A) the signal depended sigmoidally on the concentration of 2OG. The sigmoids fitted a Hill number of 2, corresponding to the number of 2OG sites in the NtcA dimer [15,17], indicating cooperativity between both sites for 2OG binding. The 2OG concentrations needed for half-maximal increase in the resonance signal differed for the three promoter fragments tested (Fig.…”

“…2OG activates NtcA-dependent gene expression regulation [10,11] and also triggers (when ATP is also bound) the release of PipX from PII [13], making PipX available for coactivation of NtcA. The structures of NtcA bound to 2OG alone [15,17] or together with PipX [15] (Fig. 1) have led to the view that PipX, by binding to each NtcA subunit, would stabilize the ''active'' conformation of NtcA that is induced by 2OG [15].…”

Structures of complexes comprised of Fischerella transcription factor HetR with Anabaena DNA targets

“…There are some clues to the biochemical mechanisms involved in differentiation. Two proteins, PII and NtcA, respond to the relative levels of glutamine and 2-oxoglutarate (2OG) (5)(6)(7). When 2OG is high, signaling nitrogen starvation, PII and NtcA activate transcription of NrrA (8), which in turn activates transcription of another protein, HetR, which also functions as a transcription activator.…”

A Novel Mechanism, Linked to Cell Density, Largely Controls Cell Division in Synechocystis

“…Synechocystis can take up and metabolize different nitrogen sources (nitrate, nitrite, ammonium, urea, and some amino acids; Quintero et al, 2000;Valladares et al, 2002;Muro-Pastor et al, 2005). Intriguingly, the control of the expression and activity of transporters and enzymes related to nitrogen metabolism is regulated by a complex network involving 2-oxoglutarate, NtcA (nitrogen control factor of cyanobacteria), PII (nitrogen regulatory protein PII), and PipX (PII-interacting protein X; Alfonso et al, 2001;Herrero et al, 2001;Llácer et al, 2010;Zhao et al, 2010;Espinosa et al, 2014;Lüddecke and Forchhammer, 2015).…”