The PipX Protein, When Not Bound to Its Targets, Has Its Signaling C-Terminal Helix in a Flexed Conformation.

ABSTRACT

PipX, an 89-residue protein, acts as a coactivator of the global nitrogen regulator NtcA in cyanobacteria. NtcA-PipX interactions are regulated by 2-oxoglutarate (2-OG), an inverse indicator of the ammonia abundance, and by PII, a protein that binds to PipX at low 2-OG concentrations. The structure of PipX, when bound to NtcA or PII, consists of an N-terminal, five-stranded ß-sheet (conforming a Tudor-like domain), and two long α-helices. These helices adopt either a flexed conformation, where they are in close contact and in an antiparallel mutual orientation, also packing against the ß-sheet, or an open conformation (observed only in the PII-PipX complex) where the last α-helix moves apart from the rest of the protein. The aim of this work was to study the structure and dynamics of isolated PipX in solution by NMR. The backbone chemical shifts, the hydrogen-exchange, and the NOE patterns indicated that the isolated, monomeric PipX structure was formed by an N-terminal five-stranded ß-sheet and two C-terminal α-helices. Furthermore, the observed NOEs between the two helices, and of α-helix2 with ß-strand2 suggested that PipX adopted a flexed conformation. The ß-strands 1 and 5 were highly flexible, as shown by the lack of interstrand backbone-backbone NOEs; in addition, the 15N-dynamics indicated that the C terminus of ß-strand4 and the following ß-turn (Phe42-Thr47), and the C-cap of α-helix1 (Arg70-Asn71) were particularly mobile. These two regions could act as hinges, allowing PipX to interact with its partners, including PlmA in the newly recognized PII-PipX-PlmA ternary complex.