Structure of the complex formed between Af1521 and ADP-ribose. (A) The ADP-ribose molecule binds the Af1521 macro domain in an L-shaped cleft. The ADP-ribose ligand is shown as a ball-and-stick model. (B) Structure of the complex between Af1521 and ADP. The structure is highly similar to that of the complex between Af1521 and ADP-ribose, but a number of interactions that contribute to ADP-ribose specificity and affinity cannot occur. The ADP ligand is shown as a ball-and-stick model.

The macro domain ligand binding pocket is selective for ADP-ribose. Isothermal titration calorimetry profiles for the binding of (A) ADP, (B) AMP and (C) NAD to the Af1521 macro domain protein. Reaction conditions were identical to those of Figure 2. The graphs clearly show decreased affinities of the macro domain for these nucleotide ligands compared to ADP-ribose (Figure 2).

High-affinity binding of the macro domain to ADP-ribose. Isothermal titration calorimetry profile: Titration of ADP-ribose ligand into a solution containing the purified Af1521 macro domain. The asterisk denotes the first, small volume injection that is not used for subsequent data fitting. The inset shows the fit of the data to an equilibrium binding isotherm. The fit provides an equilibrium dissociation constant (K_D) for the binding of ADP-ribose to Af1521 of 126±21 nM.

The Af1521 and human Bal/PARP9 macro domains recognize poly-ADP-ribosylated PARP1. ³²P-labelled PAR-labelled PARP1 was incubated on nitrocellulose filter papers containing slot-blotted proteins. The membranes contained 20 pmol (lane A) and 200 pmol (lane B) of H2A, Af1521 macro domain and human Bal/PARP9 double macro domain module, as well as 200 pmol (lane A) and 400 pmol (lane B) of the control proteins lysozyme and BSA. After extensive washes, the filter membranes were dried and autoradiographed. Incubation of ³²P-NAD with the slot-blotted proteins produced no signal (data not shown). The loading of slot-blotted proteins was verified using Sypro Ruby protein blot stain.

The Af1521 macro domain hydrolyzes a phosphorylated form of ADP-ribose. Thin-layer chromatography assays of Af1521-catalyzed reaction products. Lanes (1–5) contain reaction mixtures as indicated. Lanes (6–8) contain the indicated nucleotides in buffers identical to lane 3 but without Af1521 protein and without Appr>P. The asterisk denotes a breakdown product of ADP. Abbreviations are as follows: ADP-ribose (ADPR), ADP-ribose-cyclic-1″-2″-phospate (Appr>P), ADP-ribose-1″-phosphate (Appr-1″-P), origin of the chromatographic separation (ori) and cyclic phosphodiesterase (CPDase). Assays contained 2 mM Appr>P converted to Appr-1″-P using CPDase. To this mixture, we added Af1521 macro domain protein. Following incubation, the reactions were mixed with tetrabutylammonium and run on a Alugram Nano-SIL CN/UV₂₅₄ TLC plate and using 1.5 M LiCl and 20% v/v ethanol in water. Fluorescence images were taken at 254 nm wavelength.

Specificity of the macro domain fold for ADP-ribose. (A) Electron density for the ADP-ribose ligand in the pocket of the Af1521 protein. The ADP-ribose ligand is shown as a ball-and-stick model. (B) Stereo-diagram of the ADP-ribose binding pocket. A number of critical interactions between the ligand and the Af1521 macro domain are shown. Several of the interactions involve hydrogen bonds between side chains (Asn 34, Asp 20) and backbone amide bonds. Specific aromatic stacking interactions occur between Tyr 176 and the adenine base. The phosphates are stabilized by a number of interactions, including the backbone amide of Val 43, Ser 141 and the favorable dipole of helix 1. (C) Schematic representation for the binding of ADP-ribose to the macro domain. The LigPlot (Wallace et al, 1995) diagram summarizes key noncovalent interactions between the ADP-ribose ligand and the Af1521 macro domain. Legend: thick blue lines, ADP-ribose ligand; thin red lines, macro domain residues; circles or semicircles with radiating lines; atoms or residues involved in hydrophobic contacts between protein and ligand.

ADP-ribose binding is a conserved feature among macro domains. (A) Sequence and structure conservation in the macro domain family of proteins. Regions with the highest sequence conservation among macro domain proteins are shown in blue. These include residues (19–22), (29–34), (40–43), (98–103), (136–146) and 176. (B) Structure-guided alignment between select macro domain proteins. For the purpose of this alignment, only the macro domains used in this study were aligned. The alignment was generated using the output of a Blast search, and refined manually on the Af1521–ADP-ribose complex structure. Residues shown in blue correspond to the region colored in blue of panel (A). (C) Pulldown assays for the binding of ADP-ribose to yeast and human macro domains. Distinct macro domain proteins (A. fulgidus Af1521, S. cerevisiae YBR022W, human Alc1, human macroH2A and human BAL/PARP9) were fused to either GST protein or to a histidine-tag. In all, 30 nmol of fusion proteins was immobilized to a solid support, including two control proteins that should not interact with ADP-ribose (GST and a GST-fusion of the TAF_II250 (hTAF1) double bromodomain module (Jacobson et al, 2000)), and incubated with 30 nmol of ADP-ribose in solution. Following the incubation, the samples were centrifuged and the amount of ADP-ribose that remained in the supernatant was estimated by absorbance measurements. The graphs show the percentage retention on the beads (calculated by subtracting the percentage of ADP-ribose that remained in the supernatant from 100%). The pulldown assay shows that all tested macro domain proteins retain some ADP-ribose.