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.

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADP- ribose modifications

“…The copyright holder for this preprint (which this version posted May 19, 2021. ; https://doi.org/10.1101/2021.05. 19.444852 doi: bioRxiv preprint 4 by blocking NAD + binding to the active site 11 . A BRCT domain is located adjacent to an extended linker region that bears the primary sites for PARP1 automodification.…”

“…HPF1 was also shown to reduce PARP1 catalytic output, to decrease the length of PAR formed by PARP1 and PARP2, and to promote trans ADP-ribosylation of histones in relation to cis modification of PARP1 itself 17 . Crystal structures of HPF1 bound to the PARP2 CAT domain lacking an HD 18 , or to the PARP1 CAT domain lacking an HD 19 , have revealed the mechanistic basis for HPF1 effect on PARP1 and PARP2. HPF1 binds to PARP1 and PARP2 CAT and inserts a Glu residue to complement the active site (Fig.…”

“…By comparison to the active site of cholera toxin-like ADP-ribosyl transferases, PARP1 and PARP2 are deemed to lack a second Glu residue, in addition to the catalytic Glu of the conserved His-Tyr-Glu triad motif (Glu988 in PARP1 and Glu545 in PARP2) 18 . The second Glu is provided by HPF1 and deprotonates Ser residues to allow their ADP-ribosylation 18,19 . In was not certified by peer review) is the author/funder.…”

“…The copyright holder for this preprint (which this version posted May 19, 2021. ; https://doi.org/10.1101/2021.05. 19.444852 doi: bioRxiv preprint 5 contrast to Ser, Glu and Asp are naturally deprotonated at neutral pH and therefore do not require HPF1 to be ADP-ribosylated by PARP1. In further support, a recent cryo-electron microscopy (EM) structure of a PARP2-HPF1 complex bound to nucleosomes provided a snapshot of how PARP2 assembles with HPF1 on a DNA break in the context of chromatin 20 .…”

“…Structural analysis has highlighted a 1:1 complex of HPF1 with PARP1/2 ready to perform ADP-ribose modification of Ser 18,19 . Moreover, PARP1 modification of Ser residues is the predominant form of PARylation performed by PARP1 in response to DNA damage 21 .…”