Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a distinctive mode of engaging poly(ADP-ribose)

Abstract

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function.

Keywords: ADP-ribose; ATP; CP: Molecular biology; PARP13; SEC-SAXS; WWE domain; X-ray crystallography; ZAP; fluorescence polarization; poly(ADP-ribose).

Figure 1.. Crystal structure of mP13 ZnF5-WWE1-WWE2 bound to ADPr

(A) Domain organization of human PARP13 (hP13)/ZAP. The ZnF5-WWE1-WWE2 residue boundaries for hP13 are 507–699 and for mP13 are 476–673. (B) Crystal dimer. The crystallographic asymmetric unit contains two molecules of mP13 ZnF5-WWE1-WWE2 (red, molecule A; green, molecule B). Both molecules have an ADPr molecule bound to WWE2. Residues C488 and C496 form part of ZnF5, which is partially ordered in this structure. (C) Crystal monomer. One ZnF5-WWE1-WWE2 molecule from the asymmetric unit is shown, with the extended linker (purple), WWE domains (green), ZnF5 (deep blue), and residues leading to the catalytic domain (gray) labeled. (D) Compact monomer. Crossing over between the linker regions of the two protein molecules (small dashed line labeled “linker”) models a compact arrangement of WWE1 and WWE2 from different chains (compact monomer in blue). (E) Model for the compact monomer with the modified linker in purple, the WWE domains in green, ZnF5 region in deep blue, and residues leading to the CAT domain in gray.

Figure 2.. Biophysical analysis of PARP13 ZnF5-WWE1-WWE2

(A) Alignment of the hP13 ZnF5-WWE1-WWE2 crystal structure (PDB: 7KZH) with the compact monomer model for mP13 (this study). hP13 is colored dark gray, except for the ZnF5 region in brown, and two regions that are dissimilar between the two structures are shown in green (one is the long loop in the WWE2 binding site, and the other is the linker connecting WWE1 and WWE2). mP13 is colored light gray, except for the partially ordered ZnF5 in red, and the dissimilar regions are in blue. The bound Zn atom is shown as a sphere. The inset highlights the differences in the ZnF5 conformation, the major difference between the two structures. (B) SEC-MALS analysis of ZnF5-WWE1-WWE2 from hP13 (blue) and mP13 (red) at 40 μM concentration. The light scattering data are plotted as the Rayleigh ratio (left y axis) versus elution volume from gel filtration. The mass estimation (right y axis) is shown across the indicated peaks. (C) Zn content analysis was performed on the designated samples, and the relative concentrations between samples are shown for two measurements each. Proteins were analyzed at 10 μM. A Zn concentration of 10 μM was set as 100, such that the values for protein samples represent the percent of molecules with bound Zn. hP13 and mP13 refer to the ZnF5-WWE1-WWE2 fragment. RNF-146 is a control protein that does not contain a Zn finger, ZnF3 of hP1 is a control protein that does contain a Zn finger, and the dialysis buffer was used to estimate background levels of Zn.

Figure 3.. Ligand-binding properties of the WWE2 domain

(A) ATP bound within the cavity of WWE2. A weighted 2F_O-F_C electron density map is shown around the ATP molecule and is contoured to the 1.5σ level. (B) ADPr bound within the cavity of WWE2. A weighted 2F_O-F_C electron density map is shown around the ADPr molecule and is contoured to the 1 σ level. (C) A view of key WWE2 residues interacting with ADPr. Interatomic distances (Å) are indicated next to dashed lines connecting atoms that make close contacts. (D) Structure of WWE1 (blue/gray) superimposed on WWE2/ADPr of mP13 (green/gray). The similar structural features are colored gray, whereas the unique features are color coded. (E) Surface representation of WWE1 (on left in blue) illustrating the lack of a binding cavity compared with WWE2 (on right in green) with a deep cleft forming the ADPr binding pocket. (F) Structure of the WWE domain of RNF-146 bound to iso-ADPr (PDB: 3V3L) in pink/gray superimposed on WWE2 of mP13 bound to ADPr (green/gray). (G) Surface representation of WWE from RNF-146 bound to iso-ADPr (pink) superimposed on WWE2 of mP13 bound to ADPr (green).

Figure 4.. Biophysical and biochemical analysis of mP13 and hP13 ZnF5-WWE1-WWE2

(A) FP binding assay using ATP-FAM and the indicated proteins. A 1:1 binding model was fit to the data (solid line, hP13 wild type [WT]). These experiments were repeated three times to provide an average K_D value and associated standard deviation for hP13 WT of 97.1 ± 10.4 nM, whereas hP13 W611A and RNF-146 showed no indication of binding to ATP-FAM. The data points represent the mean polarization values and standard deviations for the three repeats of each experiment. (B) DSF analysis of relative thermal stability for the indicated proteins at 8 μM concentration in the absence of ligand or in the presence of ATP or ADPr, as indicated. The two repeats of the experiment are plotted. (C) Delta T_M (ΔT_M) comparison from DSF analysis performed with hP13 ZnF5-WWE1-WWE2 at 8 μM concentration in the absence/presence of ADPr or iso-ADPr at the indicated concentrations. The individual data points are shown for the four repeats of the experiment. The bar represents the mean value, and the error bars represent standard deviations.

Figure 5.. PAR-binding properties of ZnF5-WWE1-WWE2

(A) Illustration of a PAR ligand used in this study with the termini labeled. The ribose-ribose linkage of PAR is denoted by the 2′ to 1″ linkage. The 2′ end of PAR was labeled on the 2′ OH using the ELTA method, and the 1″ end was labeled on the terminal phosphate, labeled with an asterisk. (B) Apparent K_D values determined from FP competition assays using ADPr and the indicated PAR chains. The competitor ligands were tested for their capacity to outcompete ATP-FAM probe binding to hP13 ZnF5-WWE1-WWE2 (see Figure S4A). The individual K_D values from experiment repetitions are shown. The bar represents the mean K_D, and the error bars represent standard deviations. (C and D) Representative curves from PAR-binding analysis using PAR FAM labeled on the 1″ end (C) or using PAR FAM labeled on the 2′ end (D). A 1:1 binding model was fit to the data (solid lines). The experiments were repeated at least three times to produce the average K_D values and the associated standard deviations shown in (C) and (D). See Figures S4B and S4C for all binding curve replicates.

Figure 6.. Evidence for a PAR binding groove

(A) Electrostatic surface potential of hP13 ZnF5-WWE1-WWE2 using crystal structure PDB: 7KZH. The putative binding groove is highlighted with units of PAR, represented as green bars, tracing along the groove on the surface of WWE1 and WWE2. The terminal ADPr in the binding pocket is designated n, and the preceding ADPr units are designated n−1, n−2, and so forth, providing a rough estimate of the distance between the WWE2 binding pocket and the deepened region of the groove. The inset panel shows features of the mP13 crystal structure surrounding the bound phosphate ion, and the hP13 structure is aligned to mP13. (B) Fluorescence polarization binding affinities measured using ATP-FAM for the indicated proteins. (C) Fluorescence polarization binding affinities measured using fluorescent PAR FAM labeled on the 1″ end for the indicated proteins. For (B) and (C), the individual K_D values are plotted. The bar represents the mean values, which are also listed on the plot. The error bars represent standard deviations, and they are also listed on the plot