A New P_II Protein Structure Identifies the 2-Oxoglutarate Binding Site

Abstract

P_II proteins of bacteria, archaea, and plants regulate many facets of nitrogen metabolism. They do so by interacting with their target proteins, which can be enzymes, transcription factors, or membrane proteins. A key feature of the ability of P_II proteins to sense cellular nitrogen status and to interact accordingly with their targets is their binding of the key metabolic intermediate 2-oxoglutarate (2-OG). However, the binding site of this ligand within P_II proteins has been controversial. We have now solved the X-ray structure, at 1.4 Å resolution, of the Azospirillum brasilense P_II protein GlnZ complexed with MgATP and 2-OG. This structure is in excellent agreement with previous biochemical data on 2-OG binding to a variety of P_II proteins and shows that 2-oxglutarate binds within the cleft formed between neighboring subunits of the homotrimer. The 2-oxo acid moiety of bound 2-OG ligates the bound Mg²⁺ together with three phosphate oxygens of ATP and the side chain of the T-loop residue Gln39. Our structure is in stark contrast to an earlier structure of the Methanococcus jannaschii GlnK1 protein in which the authors reported 2-OG binding to the T-loop of that P_II protein. In the light of our new structure, three families of T-loop conformations, each associated with a distinct effector binding mode and characterized by a different interaction partner of the ammonium group of the conserved residue Lys58, emerge as a common structural basis for effector signal output by P_II proteins.

Introduction

P_II proteins are involved in the regulation of many aspects of nitrogen metabolism.¹ They can be regarded as signal integration proteins whose output is their signal-dependent interaction with various target proteins that they may activate or inhibit.2, 3, 4 The universally conserved and probably most ancient signals controlling P_II activities are the effector molecules ATP, ADP, and 2-oxoglutarate (2-OG). Additionally, many P_II proteins are covalently modified by enzymes whose activities are regulated by another key nitrogen metabolite, glutamine. P_II proteins are compact, cylindrically shaped (homo)trimers composed of 12-kDa to 13-kDa subunits from which three long exposed loops, the so-called T-loops, protrude (Fig. 1), as first reported for Escherichia coli GlnB.⁵ The T-loops are significantly conserved in sequence, but as is apparent from the numerous reported structures of P_II proteins, they are structurally very flexible.3, 6 They are vital for P_II interactions with many of their targets and are also the sites of reversible covalent modification. In addition to the T-loops, the highly conserved structure of P_II proteins is characterized by three lateral intersubunit clefts within which two smaller loops (the B-loops and C-loops) from opposing subunits contribute to an adenyl-nucleotide binding pocket where ADP or ATP can bind competitively (Fig. 1).

The binding mode of the other key effector 2-OG, which is assumed to be conserved among all P_II proteins, has remained enigmatic and controversial.³ Here we report a high-resolution structure of a complex of the P_II protein GlnZ from Azospirillum brasilense complexed with the effectors ATP and 2-OG. GlnZ and its orthologues are specifically involved in the regulation of nitrogenase activity in some nitrogen-fixing bacteria.7, 8 The observed 2-OG binding site is in excellent agreement with biochemical data, in contrast to a previously reported binding mode.⁹ This new structure will greatly facilitate an understanding of the link between changes in cellular effector pools and P_II signaling.