Crystal structure of the GlnZ-DraG complex reveals a different form of P_II-target interaction

Abstract

Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the P II family. P II proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to P II and also by P II posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the P II protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known P II complexes, the majority of contacts with the target protein do not involve the T-loop region of P II . Hence this structure identifies a different mode of P II interaction with a target protein and demonstrates the potential for P II proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZDraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.N itrogen is an indispensable element for life. In bacteria and plants, many aspects of the regulation of nitrogen metabolism are controlled by a class of ubiquitous proteins called P II signal transduction proteins (1-4). P II proteins are present in all domains of life and are one of the most widely distributed signal transduction proteins in nature: some prokaryotes encode multiple P II homologues (2, 3). P II proteins act as a central processing unit receiving signals of carbon, energy, and nitrogen levels, integrating and transforming them into different P II conformational states. The multitude of P II conformations control their ability to interact with, and thus regulate the activity of, a variety of target proteins including transporters, enzymes, and transcriptional regulators (4). P II proteins form compact barrel-shaped homotrimeric structures, in which each monomer is 12-14 kDa containing three functionally important loops: the T-, B-, and C-loops (4). The 20-residue long T-loop is structurally very flexible and participates in protein interactions in all the structures of P II -target complexes solved to date. The T-loop also contains the site of posttranslational modification if it is needed. In Proteobacteria, Y51 in the T-loop is subjected to reversible uridylylation in response to the cellular glutamine levels (5). The effector molecules ATP, ADP, and 2-oxoglutarate (2-OG) influence the conformational states of P II pr...

Influence of the ADP/ATP ratio, 2-oxoglutarate and divalent ions on Azospirillum brasilense PII protein signalling

“…This interaction is abolished when the GlnZ binding sites are empty or when bound only to ATP (Huergo et al, 2009;Rajendran et al, 2011). A similar response has been observed for the AmtB-GlnZ and AmtB-GlnB complexes (Rodrigues et al, 2011).…”

“…The A. brasilense GlnD, DraT, DraG and AmtB proteins were expressed and purified as N-terminally 66His-tagged proteins, as described previously (Huergo et al, 2007(Huergo et al, , 2009). The A. brasilense GlnB, GlnZ, GlnZ Q39K and GlnZ Q39E proteins were expressed and purified as described previously (Moure et al, 2012;Rajendran et al, 2011). The GlnZ Q39E mutant was obtained using the Quick-Change site-directed mutagenesis kit (Agilent) using the pMSA4 plasmid (Moure et al, 2012) as a template.…”

The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB

“…S2 in the supplemental material). Furthermore, the deletion of residues 42 to 54 in the highly similar GlnZ protein did not affect binding of ATP and ADP (24), supporting the conclusion that the GlnB⌬42-54 variant retains its overall fold and effector binding properties. Comparison of the GlnB and GlnZ T-loop sequences revealed only 3 nonconserved substitutions at positions 42, 52, and 54 (see Fig.…”

“…At the same time, GlnB and GlnZ are deuridylylated (20) and interact with DraT and DraG, respectively (21)(22)(23)(24). The DraG-GlnZ complex is directed to the membrane by interaction with the ammonium transporter AmtB, resulting in DraG inactivation (20,22,24). Given that the DraT-GlnB complex is detected only under conditions where DraT is active, it was postulated that this protein-protein interaction is responsible for DraT activation; however, this has yet to be confirmed (23).…”

“…Upon an ammonium pulse, nitrogenase is inactivated by ADP-ribosylation of the Fe protein. At the same time, GlnB and GlnZ are deuridylylated (20) and interact with DraT and DraG, respectively (21)(22)(23)(24). The DraG-GlnZ complex is directed to the membrane by interaction with the ammonium transporter AmtB, resulting in DraG inactivation (20,22,24).…”