The First Residue of the PWWP Motif Modulates HATH Domain Binding, Stability, and Protein–Protein Interaction

Abstract

Hepatoma-derived growth factor (hHDGF) and HDGF-related proteins (HRPs) contain conserved N-terminal HATH domains with a characteristic structural motif, namely the PWWP motif. The HATH domain has attracted attention because of its ability to bind with heparin/heparan sulfate, DNA, and methylated histone peptide. Depending on the sequence of the PWWP motif, HRP HATHs are classified into P-type (Pro-His-Trp-Pro) and A-type (Ala-His-Trp-Pro) forms. A-type HATH is highly unstable and tends to precipitate in solution. We replaced the Pro residue in P-type HATHHDGF with Ala and evaluated the influence on structure, dynamics, and ligand binding. Nuclear magnetic resonance (NMR) hydrogen/deuterium exchange and circular dichroism (CD) measurements revealed reduced stability. Analysis of NMR backbone (15)N relaxations (R1, R2, and nuclear Overhauser effect) revealed additional backbone dynamics in the interface between the β-barrel and the C-terminal helix bundle. The β1-β2 loop, where the AHWP sequence is located, has great structural flexibility, which aids HATH-HATH interaction through the loop. A-type HATH, therefore, shows a stronger tendency to aggregate when binding with heparin and DNA oligomers. This study defines the role of the first residue of the PWWP motif in modulating HATH domain stability and oligomer formation in binding.

Role of hepatoma‐derived growth factor in promoting de novo lipogenesis and tumorigenesis in hepatocellular carcinoma

“…HDGF is a unique growth factor and may act through either a receptor‐mediated pathway or a more direct way, that is, DNA, RNA, or protein binding(Chen et al ., 2018; Hung et al ., 2015; Rona et al ., 2016). We found that HDGF mainly colocalized with SREBP‐1a within the nucleus, implying that nHDGF rather than receptor‐bound HDGF involved in SREBP‐mediated transcription (Fig.…”

“…4A). HDGF contains a conserved PWWP domain previously known as the HATH domain, which is required for binding with heparin, DNA, and protein (Chen et al ., 2018; Hung et al ., 2015; Rona et al ., 2016). According to the first amino acid, the PWWP domain is classified into P‐type (Pro–His–Trp–Pro) and A‐type (Ala–His–Trp–Pro).…”

“…According to the first amino acid, the PWWP domain is classified into P‐type (Pro–His–Trp–Pro) and A‐type (Ala–His–Trp–Pro). Structural analysis reveals that A‐type HDGF mutant (P24A) apparently impact its ability in DNA binding and protein–protein interaction (Hung et al ., 2015). Therefore, we mutated the Pro residue in P‐type HDGF (WT) to Ala (P24A mutant) and evaluated the effect on gene expression.…”

“…The PWWP domain was first characterized from the WHSC1 (Wolf–Hirschhorn syndrome candidate 1) gene and was previously known as the HATH domain (Bao et al ., 2014b). Proteins that contain the PWWP domain are involved in transcriptional regulation, DNA methylation, histone modification, and DNA repair by interacting with histone, double‐stranded DNA, and negatively charged molecules such as heparin (Chen et al ., 2018; Hung et al ., 2015; Rona et al ., 2016). The type of PWWP domain modulates its binding and protein–protein interaction (Hung et al ., 2015).…”

“…HDGF is a member of a family of growth factors called HDGF‐related proteins, consisting of a highly conserved N‐terminal 100‐residue PWWP domain, also called the HATH domain (homologous to the N terminus of HDGF) and a disordered C‐terminal 140‐residue domain (Chen et al ., 2015a). Recent evidence indicates that the first residue of the PWWP motif in HDGF regulates the PWWP domain structure, protein stability, and protein–protein interactions (Hung et al ., 2015). HDGF is a broad regulator of cancer cell activity and is involved in many cellular processes including transformation, apoptosis, angiogenesis, and metastasis (Bao et al ., 2014b).…”