Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII

Abstract

PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

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

“…Furthermore, this affinity is importantly increased by PipX, which is confirmed to bind only to NtcA if 2OG is present, supporting the view [15] that PipX stabilizes the ''active'' 2OG-bound conformation of NtcA. Finally, titration of PipX binding to NtcA strongly suggests that the affinity of PipX for this transcription factor is much higher than that reported earlier for protein PII [15]. [15,29,30].…”

“…Finally, titration of PipX binding to NtcA strongly suggests that the affinity of PipX for this transcription factor is much higher than that reported earlier for protein PII [15]. [15,29,30]. Right, cAMP activation of CRP.…”

“…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].…”

“…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].…”

“…1). Crystal structures for the PII-PipX [15,16] Abbreviations: NtcA, nitrogen control factor of cyanobacteria; CRP, cAMP receptor protein, also called CAP, catabolite gene activator protein; PipX, PIIinteracting protein X; SYCRP1, Synechocystis CRP; 2OG, 2-oxoglutarate; RU, relative unit; SPR, surface plasmon resonance; FC1 and FC2, flow cells 1 and 2, respectively of the Biacore chip; IPTG, isopropyl-b-D-thiogalactoside only binds to PII if MgATP also binds), three PipX molecules are sequestered by one PII trimer in such a way that they become unavailable for activating NtcA. 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.…”