Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Abstract

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

Structure of human DPPA3 bound to the UHRF1 PHD finger reveals its functional and structural differences from mouse DPPA3

“…Thus, we identified the corresponding region of hDPPA3 by sequence alignment (residues 81-118 [hDPPA3 81-118 ] (Figure 1a and 1b), and evaluated whether this region binds to the human UHRF1 PHD finger, residues 299–366 (hPHD). Isothermal titration calorimetry (ITC) demonstrated that hDPPA3 81-118 could bind to hPHD with a K d of 0.868 μM (Figure 1c), which is approximately 30-fold weaker than the binding affinity between mDPPA3 and mPHD ( K d = 0.0277 μM) 29 . The binding affinity of hDPPA3 to hPHD 81-118 is comparable with the previously reported binding affinity between hPHD and the histone H3 N-terminal tail (residues 1–15; K D = 1.7 μM) or PAF15 (residues 1–10; K D = 2.2 μM) 17,31 .…”

“…Our previous NMR structural analysis of mDPPA3 complexed with mUHRF1 PHD (mPHD) revealed that residues 85–118 of mDPPA3 are essential for its interaction with mPHD (Figure 1b and 2b) 29 . Thus, we identified the corresponding region of hDPPA3 by sequence alignment (residues 81-118 [hDPPA3 81-118 ] (Figure 1a and 1b), and evaluated whether this region binds to the human UHRF1 PHD finger, residues 299–366 (hPHD).…”

“…However, it is unclear if the biological function of mDPPA3 is conserved in human DPPA3 (hDPPA3), and its role in human oocytes and preimplantation embryos is unknown. Two α-helices in mDPPA3 which are induced upon binding to mPHD has been shown to be required for the interaction with mUHRF1 29 . However, the amino acid sequences corresponding to these helices are poorly conserved between human and mouse DPPA3 (Figure 1a), which raises a question of whether hDPPA3 also binds to the hUHRF1 PHD finger in a manner similar to their mouse counterparts, and whether hDPPA3 can inhibit chromatin binding of UHRF1.…”

“…mDPPA3 plays an important role in the formation of oocyte-specific DNA methylation patterns by preventing excessive de novo DNA methylation mediated by UHRF1 24 . Using nuclear magnetic resonance (NMR) solution structural analysis of mouse the UHRF1 PHD finger (mPHD) bound to mDPPA3, we recently revealed that the C-terminal region of mDPPA3 binds to mPHD utilizing a VRT motif at residues 88–90: 88 VRT 90 , which is conserved in the motifs of other binding partners, histone H3 1 ART 3 and PAF15 1 VRT 3 with two subsequent α-helices unique to mDPPA3 (Figure 2b) 29 . Owing to this multifaceted interaction, the binding affinity of mDPPA3 to mPHD ( K D of 0.0277 μM) is significant stronger than those of histone H3 and PAF15 ( K D of 1.59 μM and 3.52 μM, respectively), indicating that the mechanism by which mDPPA3 inhibits chromatin-binding of UHRF1 involves the competitive binding of between mDPPA3 and histone H3/PAF15 to UHRF1 29 .…”

Nondomain biopolymers: Flexible molecular strategies to acquire biological functions

“…DPPA3/Stella/Pgc7, an intrinsically disordered protein without a recognizable functional domain, forms a tight complex with UHRF1, which regulates the maintenance of DNA methylation along with Dnmt1 (Li et al, 2018). DPPA3 undergoes a “disorder‐to‐order” structural transformation and tightly associates with the PHD domain of UHRF1 (Hata et al, 2022). This interaction results in the dissociation of UHRF1 from histones and subsequent nuclear export of this chromatin modifier.…”