Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Li, Xiao Dan; Huergo, Luciano F.; Gasperina, Antonietta; Pedrosa, Fábio O.; Merrick, Mike; Winkler, Fritz K.

doi:10.1016/j.jmb.2009.05.031

Cited by 16 publications

(16 citation statements)

References 29 publications

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“…The T-loop of GlnZ (residues 37-55) is only ordered at its base (up to K40 and again from V53). The metal binding sites at the active site of DraG are occupied by two Mg 2þ ions as observed in the previous structure of DraG [PDB ID 3G9D, (25)]. …”

Section: Resultsmentioning

confidence: 82%

“…The GlnZ-DraG complex reconstituted in vitro was purified by gel filtration chromatography and crystallized in the presence of ADP. Crystals diffract to 2.1 Å and the structure solution was performed by molecular replacement using MOLREP within the ccp4i suite (24) using as search models GlnZ -PDB ID 3MHY (7) and DraG -PDB ID 3G9D (25).…”

Section: Resultsmentioning

confidence: 99%

“…Alignment of the DraG and P II amino acid sequences (Fig. S4) and comparison of the A. brasilense (25) and R. rubrum DraG crystal structures (30) revealed that many contacts mapped in the A. brasilense GlnZ-DraG complex, are predicted to be conserved in the R. rubrum GlnJ-DraG complex (Table S1). In only a few cases there are changes in one or both proteins that could enable modified contacts.…”

Section: Comparison Of Glnz-drag Complex With the Putative Glnj-dragmentioning

confidence: 99%

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

Rajendran

Gerhardt

Bjelić

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Comparison Of Glnz-drag Complex With the Putative Glnj-dragmentioning

confidence: 99%

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

Rajendran

Gerhardt

Bjelić

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…The DraG‐GlnZΔ42‐54 complex was crystallized and the X‐ray structure was determined at 1.55 Å resolution ( R work = 14.4%, R free = 16.5%) by molecular replacement using the DraG‐GlnZ complex as search model. The structure shows no significant changes in the DraG structure or the DraG‐GlnZ interaction interface compared to the previously published structures (Table and Fig. ).…”

Section: Resultsmentioning

confidence: 39%

The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense

et al. 2019

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The ammonium‐dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADP‐ribosyl transferase (DraT) and dinitrogenase reductase ADP‐glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP‐ribosylated Fe‐protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG‐GlnZ‐AmtB complex in vitro. Previous structural data have revealed that when the DraG‐GlnZ complex associates with AmtB, the flexible T‐loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG‐GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T‐loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T‐loop (GlnZΔ42‐54). However, although the GlnZΔ42‐54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild‐type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG‐GlnZ‐AmtB is necessary for the inactivation of DraG.

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“…Crystal structures of DRAG from two different bacteria, Rhodospirillum rubrum and Azospirillum brasilense (Berthold et al, 2009; Li et al, 2009), show extensive structural homology with the ARH enzymes. The overall structures are compact and purely α-helical, and the active sites are located in a deep cleft surrounded by loops, Fig.…”

Section: Introductionmentioning

confidence: 99%

Structural biology of the writers, readers, and erasers in mono- and poly(ADP-ribose) mediated signaling

Karlberg

Langelier

Pascal

et al. 2013

Molecular Aspects of Medicine

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ADP-ribosylation of proteins regulates protein activities in various processes including transcription control, chromatin organization, organelle assembly, protein degradation, and DNA repair. Modulating the proteins involved in the metabolism of ADP-ribosylation can have therapeutic benefits in various disease states. Protein crystal structures can help understand the biological functions, facilitate detailed analysis of single residues, as well as provide a basis for development of small molecule effectors. Here we review recent advances in our understanding of the structural biology of the writers, readers, and erasers of ADP-ribosylation.

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Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Cited by 16 publications

References 29 publications

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

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

The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense

Structural biology of the writers, readers, and erasers in mono- and poly(ADP-ribose) mediated signaling

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Crystal Structure of Dinitrogenase Reductase-activating Glycohydrolase (DRAG) Reveals Conservation in the ADP-Ribosylhydrolase Fold and Specific Features in the ADP-Ribose-binding Pocket

Cited by 16 publications

References 29 publications

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

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

The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense

Structural biology of the writers, readers, and erasers in mono- and poly(ADP-ribose) mediated signaling

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

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