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.

Fig. 1.

Posttranslational modification of proteins by reversible ADP-ribosylation. Mono- and poly-ADP-ribosyl transferases (ARTs and PARPs) catalyze the transfer of an ADP-ribose moiety from β-NAD⁺ to a target amino acid side chain or previously linked ADP-ribose on a protein, releasing nicotinamide in the process. The reverse reaction is catalyzed by mono- and poly-(ADP-ribosyl) glycohydrolases (ARHs and PARGs), that cleave the α-glycosidic bond between C1′ and the protein side chain or the preceding ADP-ribose.

Fig. 2.

Overall structure of DraG and dinuclear Mn site. (A) Cartoon representation of the monomeric DraG holoenzyme colored in rainbow. (B) Close up of the dimanganese site. Manganese ions are colored in mangenta and surrounding residues in cyan. The unidentified ligand, modeled as a formate, is shown in yellow. Green lines indicate metal coordination and the dashed green line a hydrogen bond. Coordination distances in Ångström.

Fig. 3.

Complex structures of DraG. (A) The reaction intermediate analogue structure with ADP-ribosyllysine on the surface of one DraG monomer reaching into the active site of another monomer. Inset: Amino acids with side chains involved in the binding site displayed as sticks. (B) 2Fo-Fc map for the ADP-ribosyllysine contoured at 1.0 σ. (C) 2Fo-Fc map for ADP-ribose in the D97N variant structure contoured at 1.0 σ. (D) Stereoview of the superimposed open ADP-ribosyllysine (yellow) and closed α-ADP-ribose (gray) in the active site. Interacting amino acids are shown in stick representation in dark green with red dashed bonds for the ADP-ribosyllysine complex structure and in pink with gray dashed bonds for the ADP-ribose D97N complex structure. Mn_A is shown in magenta. Ribose atoms are labeled in black for the closed ribose and in red for the open conformation.

Fig. 4.

Immunoblot of dinitrogenase reductase showing the enzymatic activity of wild-type DraG and variants. The upper band indicates ADP-ribosylated dinitrogenase reductase, and the lower band represents the un-modified form. The starting material contained the homodimeric dinitrogenase reductase with one of the subunits ADP-ribosylated, giving rise to the double bands observed. Complete hydrolysis of ADP-ribosylated dinitrogenase reductase results in a single lower band. (A) The reactions were carried out without DraG enzyme added (the control lane) or with indicated enzyme variants. The reactions were quenched after 16 h of incubation when full de-ADP-ribosylation was expected even for very low activities. (B) Activities of the Glu-28 variants were assayed in a time-course experiment and the rates of hydrolysis were compared with wild-type DraG. The reactions were quenched and samples were taken after 0, 5, 10 and 20 min.

Fig. 5.

Proposed catalytic mechanism for DraG. Amino acids actively participating in the reaction mechanism and manganese ions are displayed in gray. The circled Arg represents the ADP-ribosylated arginine in the modified protein. Dashed lines indicate hydrogen bonds and gray lines show metal coordination.