Characterization of altered regulation variants of dinitrogenase reductase‐activating glycohydrolase from <i>Rhodospirillum rubrum</i>

136

Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono-and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3D structure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-␣-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly-but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors.protein structure ͉ posttranslational modification ͉ glycohydrolase ͉ docking P osttranslational modifications (PTMs) are covalent modifications of amino acid side chains in proteins that come in many different sizes and shapes, ranging from the simple addition of a phosphate group to complex multistep glycosylations. Enzyme-catalyzed PTMs allow rapid responses to environmental stimuli and play crucial roles in signal transduction. NAD-dependent ADP-ribosylation is a reversible PTM in which mono-and polyADP-ribosyltransferases (ARTs and PARPs) and ADP-ribosylhydrolases (ARHs) and poly(ADP-ribose) glycohydrolases (PARGs) catalyze amino acid-specific ADPribosylation and de-ADP-ribosylation, respectively ( Fig. 1) (1-10). ADP-ribosylation has attracted attention because bacterial virulence factors, including diphtheria, cholera, and pertussis toxin, use it as part of their pathogenic mechanism (2, 11). Mono-and polyADP-ribosylation have been recognized also as regulatory mechanisms in many cellular processes, including DNA-repair, chromatin decondensation, transcription, telomere function, mitotic spindle formation, and apoptosis (5-10, 12).Several enzymes have been cloned that catalyze de-ADPribosylation of mono-or polyADP-ribosylated proteins (13-15). Dinitrogenase-activating glycohydrolase (DRAG), an Arg-specific ARH from the phototrophic bacterium Rhodospirillum rubrum, regulates a key enzyme of nitrogen fixation (16-18). The human genome encodes three DRAG-related proteins designated ARH1, ARH2, and ARH3 (19), which are 357, 354, and 363 residues long, respectively. ARH1, like DRAG, specifically de-ADP-ribosylates prote...

Section: Discussionsupporting

confidence: 56%

The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation

Mueller-Dieckmann

Kernstock²,

Lisurek

et al. 2006

136

“…In vivo studies showed that a R. rubrum DraG N100K variant lost regulation by ammonium, and this is predicted from our structure because the DraG K100 interaction with GlnJ R103 and the nearby R38 residues would be repulsive, thereby disfavoring complex formation (29,31). A case of two complementary changes is the interaction of A. brasilense DraG H112 with GlnZ D68 which become substituted by E112 and N68 respectively in R. rubrum permitting a similar side chain interaction.…”

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

confidence: 55%

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

Rajendran

Gerhardt

Bjelić

et al. 2011

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...

“…Visual inspection of this structure suggested further functional and structural roles for certain amino acids ( Table 1). The functional relevance of Asp and other amino acids in and near the active site has also been demonstrated experimentally by inactivating mutations of dinitrogenase reductase-activating glycohydrolase in human or rat ADP-ribosylarginine hydrolase (29)(30)(31)(32). Interestingly, most functionally relevant Asp residues are not conserved in SelJ and J1-crystallin homologues or in six other bacterial proteins (Fig.…”

Section: Selj Experimental Validationmentioning

confidence: 87%

“…It has also been shown experimentally that dinitrogenase reductase ADP-ribosyltransferases require Mg-ATP and a free divalent metal for their activity. Furthermore, a binuclear Mn 2ϩ center, also found in arginases (33), has been detected in the active site of dinitrogenase reductase-activating glycohydrolases (31)(32)(33). Therefore, the modeled Mg 2ϩ ions may really point to the approximate positions of Mg 2ϩ or Mn 2ϩ in vivo.…”

Section: Selj Experimental Validationmentioning

confidence: 99%

“…In particular, ARHs cleave ADP-ribose-L-Arg (29, 30) (EC 3.2.2.19). Other well studied family members are dinitrogenase reductase-activating glycohydrolases (EC 3.2.2.24) of the photosynthetic Rhodospirillum rubrum and related bacteria, which regulate nitrogen fixation by removing the ADP-ribosyl group of inactivated dinitrogenase reductases to recover their activity (31,32).…”

Section: Selj Experimental Validationmentioning

confidence: 99%

See 1 more Smart Citation

Diversity and functional plasticity of eukaryotic selenoproteins: Identification and characterization of the SelJ family

Castellano

Lobanov

Chapple

et al. 2005

102

Selenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. Consistent with its potential role as a structural crystallin, SelJ has preferential and homogeneous expression in the eye lens in early stages of zebrafish development. A structural role for SelJ would be in contrast to the majority of known selenoenzymes. The unusually highly restricted phylogenetic distribution of SelJ, its specialization, and the comparative analysis of eukaryotic selenoproteomes reveal the diversity and functional plasticity of selenoproteins and point to a mosaic evolution of the use of Sec in proteins.ADP-ribosylation ͉ J1-crystallins ͉ selenocysteine ͉ selenium S elenocysteine (Sec) residues are found only in a small group of proteins called selenoproteins. Selenoproteins with characterized functions are enzymes involved in redox reactions. Sec is inserted by dynamic recoding of an in-frame UGA stop codon located upstream of the actual termination codon (1). Selenoproteins are present in eukaryotes and prokaryotes and, although their sets of selenoproteins (selenoproteomes) do not completely overlap (2), their intersection appears to be larger than previously thought (3). In eukaryotes, the Sec insertion sequence (SECIS), an RNA stem-loop located in the 3Ј UTR of selenoprotein genes, recruits several transacting factors to recode UGA from termination to Sec insertion (4). The dual function of this codon confounds genefinding programs and human curators, making selenoprotein identification a challenge (5-10).In addition, Cys and Sec are structurally related amino acids, with Sec incorporating selenium in the position of sulfur in Cys. The functional resemblance of Sec and Cys is supported by the observation that their residues occupy equivalent positions in homologous proteins. As expected, mutation of the Sec residue to Cys results in variants that are active but have a lower catalytic efficiency (11-13), although they may have an enhanced translation (13). Furthermore, natural Cys-containing counterparts are usually inferior catalysts (14, 15), but similar reactivity has also been reported (16). Hence, Sec, per se, does not possess an essential role in protein function and these two residues seem to be partially interchangeable. Accordingly, Sec and Cys residues are alte...