HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

Abstract

Upon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

Fig. 1. HPF1 binds to PARP1-CAT ΔHD.

a The SEC assay indicated that HPF1 binds to PARP1-CAT ΔHD, but not full-length PARP1-CAT. b Typical ITC data where human HPF1 was titrated into a solution of PARP-1 CAT ΔHD. In this titration, the association constant (K) of HPF1/PARP-1 CAT ΔHD was estimated to be 7.58E5 ± 5.50E4 M^-1, equivalent to a dissociation constant (K_d) of ~1.3 μM. After replicated measurements, the final K_d value was determined to be 1.5 ± 0.2 μM (Table 1, Supplementary Fig. 2). c Typical ITC data where human HPF1 was titrated into a solution of full-length PARP-1 CAT. No binding was detected in this assay.

Fig. 2. Crystal structure of human HPF1/PARP1-CAT ΔHD and the binding mode between these two proteins.

a Overall structure and the interaction details between human HPF1 and PARP1-CAT ΔHD. The overall structure of human HPF1/PARP1-CAT ΔHD is shown as cartoons. HPF1 and PARP1-CAT ΔHD are colored in cyan and orange, respectively. The gray-colored molecule on the left side indicates another PARP1-CAT ΔHD molecule from a neighboring crystallographic asymmetric unit that also contacts the HPF1 molecule, shown here due to close packing of molecules in the crystal. Therefore, the complex crystal structure indicated two possible interaction modes between HPF1 and PARP1-CAT ΔHD. The three insets show the key residues on the two interfaces that mediate the interactions between the HPF1 and PARP1 in the crystal. The dashed lines indicate polar interactions between the key residues. b Mutagenesis/ADP ribosylation studies to verify which key residues indicated in the crystal structure mediate HPF1 and PARP1 binding in solution. Hyper-automodification of PARP1 (shown by the smeared PARP1^ADPr bands on SDS-PAGE) was thought to indicate a loss of the interaction between HPF1 mutants and PARP1. Surprisingly, we noted that HPF1 R239A, but not any other HPF1 mutants, was robustly ADP-ribosylated in the assay (black arrow, also see Fig. 4c), while E284A seemed to be mildly ADP-ribosylated. The HPF1 E284A band became weaker, indicating a loss of unmodified protein in the assay (gray arrow). c Amino acid sequence alignment of human PARP1/2/3. The verified key residues that mediate HPF1 binding in PARP1 in solution are indicated by red dots. The corresponding residues in PARP2 and PARP3, if conserved in PARP1, are indicated by green and blue dots, respectively.

Fig. 3. Structural basis of the mechanism through which HPF1 modulates PAPR1 activity.

a Surface electrostatic potential of human HPF1, PAPR1-CAT ΔHD and the hetero-dimer. The negatively and positively charged regions are colored red and blue, respectively. A strongly negatively charged region in HPF1 merges with the active site of PARP1-ART in the complex, creating a joint negatively charged active site. The inset on the right shows the acidic and basic residues in this negatively charged region of HPF1. b A close view of the remodeled active site and the HD domain if in the folded state. The HD domain is shown as cartoons covered by transparent surface based on a superimposition of the ART domain from the HPF1/PAPR1-CAT ΔHD structure and the ART domain from the DNA-bound PARP1 crystal structure (PDB 4DQY). The green arrow indicates the entrance to the tunnel leading to the remodeled active center. c, d Superimposition of our HPF1/PAPR1-CAT ΔHD complex structure (cyan and orange) with the previously reported PARP1-ART/BAD complex structure (PDB 6BHV, light-blue) and the PARP1-CAT/carba-NAD⁺ complex structure (PDB 1A26, lime). Two different views of the active cite are shown. BAD (light-blue sticks) is an NAD⁺ analog that mimics the ADP-ribosylation donor. The ADP moiety of carba-NAD⁺, shown as lime sticks in this figure, has been proposed to represent the ADP-ribosylation acceptor for ADPr chain elongation/branching. e A proposed catalytic mechanism of PARP1 automodification in the absence of HPF1. Without HPF1, the acidic residues in automodification domain (AD) of PARP1 can access the active center to get ADP-ribosylated. f Proposed catalytic mechanism of histone serine ADP-ribosylation in the presence of HPF1. Upon HPF1 binding, HPF1 Glu284 carboxyl is positioned approximately 4.6 Å (see the purple dashed lines in c and d) away from the donor NAD⁺ ribose (represented by BAD). This distance is too great for Glu284 itself to accept the ADP-ribose, but could catalyze ADP-ribosylation of a serine by promoting deprotonation of its sidechain hydroxyl.

Fig. 4. Depicting the function of HPF1 Arg239.

a Comparison of the HPF1/PAPR1-CAT ΔHD complex structure and the DNA-bound PARP1 crystal structure (PDB 4DQY) by superimposing the ART domains from both (orange—ART determined in this report; green—ART of PDB 4DQY). HPF1 binding to PARP1 would presumably dislodge PARP1 automodification domain (AD) that was previously proposed to be located roughly in the same position where HPF1 binds (see Supplementary Fig. 1), thus prevents automodification of PARP1. b A close view of the long α6-α7 loop region structure and its position relative to the active center of the HPF1/PARP1 complex. c Mass Spectrometry analysis revealed that HPF1 R239A mutation resulted in ADP-ribosylation on Asp235 and Glu240, a unique phenomenon only seen in this mutant (see Fig. 2B, black arrow). The mutated residue 239 is shown in blue in the sequence, while the modified residues Asp235 and Glu240 are shown in red.