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

Autores de IIS La Fe

• Martina Palomino Schätzlein

  Autor

• 

  Antonio Pineda Lucena

  Autor

• Vladimir Rubio Segarra

  Autor

Participantes ajenos a IIS La Fe

• Forcada-Nadal, A
• Neira, JL

Grupos

• Unidad de Descubrimiento de Fármacos (UDF)

  Investigador Principal

  Leonor Puchades Carrasco

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 P-II, a protein that binds to PipX at low 2-OG concentrations. The structure of PipX, when bound to Ntc.A. or P-II, consists of an N-terminal five-stranded beta-sheet (conforming a Tudor-like domain); arid two long alpha-helices. These helices adopt either a flexed conformation, where they are in close contact and in an antiparalld mutual orientation, also packing against the beta-sheet, or an open conformation (observed-onlyin the P-II PipX complex) where the last alpha-helix moves apart from the rest Ofthe 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 beta-sheet and two C-terminal alpha-helices. Furthermore, the observAd NOEs between the two helices, and of alpha-helix2 with beta-Strand2 suggested that PipX adopted a flexed conformation. The/3-strands 1 and S were highly flexible, as shown by the lack of interstrand backbone backbone NOEs; in addition, the 15N-dynamics indicated that the C terminus of beta-strand4 and the following beta-turn (Phe42-Thr47), and the C-cap of alpha-helixl (Arg70-Asn71)-were particularly mobile. These two 'regions could act as hinges, allowing PipX to interact with its partners, including PlniA in the newly recognized P-II-PipX-PlmA ternary complex.

Keywords

• N-15 NMR RELAXATION; MODEL-FREE APPROACH; MAGNETIC-RESONANCE RELAXATION; BACKBONE DYNAMICS; ROTATIONAL DIFFUSION; TIME-SCALE; P-II; 4-HELIX-BUNDLE PROTEIN; CHEMICAL-EXCHANGE; HYDROGEN-EXCHANGE