Mechanism of ADP-ribosylation removal revealed by the structure and ligand complexes of the dimanganese mono-ADP-ribosylhydrolase DraG

Abstract

ADP-ribosylation is a ubiquitous regulatory posttranslational modification involved in numerous key processes such as DNA repair, transcription, cell differentiation, apoptosis, and the pathogenic mechanism of certain bacterial toxins. Despite the importance of this reversible process, very little is known about the structure and mechanism of the hydrolases that catalyze removal of the ADP-ribose moiety. In the phototrophic bacterium Rhodospirillum rubrum, dinitrogenase reductase-activating glycohydrolase (DraG), a dimanganese enzyme that reversibly associates with the cell membrane, is a key player in the regulation of nitrogenase activity. DraG has long served as a model protein for ADP-ribosylhydrolases. Here, we present the crystal structure of DraG in the holo and ADP-ribose bound forms. We also present the structure of a reaction intermediate analogue and propose a detailed catalytic mechanism for protein de-ADP-ribosylation involving ring opening of the substrate ribose. In addition, the particular manganese coordination in DraG suggests a rationale for the enzyme's preference for manganese over magnesium, although not requiring a redox active metal for the reaction.binuclear dinuclear ͉ bioinorganic chemistry ͉ covalent modification ͉ nitrogen fixation ͉ posttranslational modifications P osttranslational modification of proteins by the attachment of chemical groups is used by cells from all kingdoms of life and occurs in several forms. Through the reversible covalent modification of specific amino acid side chains, enzyme activity, or protein function can be switched on and off as a rapid response to environmental stimuli. ADP-ribosylation is a posttranslational modification existing in two distinct forms, mono-and poly-ADPribosylation, resulting from the attachment of a single ADP-ribose moiety or a polymeric ADP-ribose chain structure, respectively, to the protein (1, 2).Mono-ADP-ribosylation was first discovered as a process used in the action of several bacterial toxins, including cholera-, diphtheria-, and pertussis toxin (3, 4). The pathogenesis of these diseases involves ADP-ribosylation of specific human proteins, catalyzed by the toxins, thereby affecting the activity of the modified proteins. In human physiology, mono-ADP-ribosylation is found in the regulation of cell-cell and cell-matrix interactions, as well as part of immune function (5, 6). Poly-ADP-ribosylation is involved in many fundamental processes such as DNA repair, transcription and cell differentiation, and is also implicated in carcinogenesis (6-8). Poly-ADP-ribosylation inhibitors are currently in phase II clinical trials for treatment of breast and ovarian cancer (9).Mono-ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs) and is specifically targeted to amino acids residues such as Arg, Asn, Glu, Asp, Cys, diphthamide, or phosphoserine. The reaction is stereospecific, using ␤-NAD ϩ as a substrate, forming the ␣-anomer of the ADP-ribosylated amino acid and releasing nicotinamide (10) (Fig. 1). PolyADP-ribosylation i...

Structures and Mechanisms of Enzymes Employed in the Synthesis and Degradation of PARP-Dependent Protein ADP-Ribosylation

“…ARH3 is structurally unrelated to PARG and is a member of the dinitrogenase reductase-activating glycohydrolase-related (DraG) protein family. DraG homologs in bacteria have been described as mono(ADP-ribosyl) hydrolases that control nitrogen fixation through protein de(ADP-ribosyl)ation (Berthold et al, 2009). ARH3, like other DraG proteins, possesses an all a-helical fold and relies on the presence of a conserved binuclear Mg 2+ cluster to assist the acid-base catalytic PAR degradation (Mueller-Dieckmann et al, 2006;Slade et al, 2011).…”

Assembly of nonheme Mn/Fe active sites in heterodinuclear metalloproteins

“…In the most basic cases a single metal ion is coordinated by protein side chains, such as in the mononuclear non-heme iron enzymes [1]. Many proteins, however, utilize large and/or multiple metal clusters, such as iron-sulfur cluster proteins [2, 3], the nitrogenase system [4–7], and cytochrome c oxidase [8, 9]. They may also include other non-protein inorganic or organic molecules, such as the hydrogenases [10, 11] or heme [12, 13] and cobalamine proteins [14].…”

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

“…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.…”