Molecular mechanism of NAD+ and NMN binding to the Nudix homology domains of DBC1

Deleted in breast cancer 1 (DBC1) is a human nuclear protein that modulates the activities of various proteins involved in cell survival and cancer progression. Oxidized form of nicotinamide adenine dinucleotide (NAD+) is suggested to bind to the Nudix homology domains (NHDs) of DBC1, thereby regulating DBC1-Poly (ADP-ribose) polymerase 1 (PARP1) interactions, resulting in the restoration of DNA repair. Using Nuclear Magnetic Resonance (NMR) and Isothermal Titration Calorimetry (ITC), we confirmed NAD+ and its precursor nicotinamide mononucleotide (NMN) both bind the NHD domain of DBC1 (DBC1354–396). NAD+ likely interacts with DBC1354–396 through hydrogen bonding, with a binding affinity (8.99 μM) nearly twice that of NMN (17.0 μM), and the key binding sites are primarily residues E363 and D372, in the agreement with Molecular Docking experiments. Molecular Dynamics (MD) simulation further demonstrated E363 and D372's anchoring role in the binding process. Additional mutagenesis experiments of E363 and D372 confirmed their critical involvement of ligand-protein interactions. These findings lead to a better understanding of how NAD+ and NMN regulate DBC1, thereby offering insights for the development of targeted therapies and drug research focused on DBC1-associated tumors. •NAD+ and NMN bound DBC1's NHD domain (DBC1354–396) at key sites E363 and D372.•NAD+ likely bonded DBC1354–396 twice as strongly as NMN via hydrogen bonds.•E363 and D372 mutagenesis confirmed their vital role in ligand-protein interaction.•DBC1354–396's N-terminal loop anchored ligands via hydrogen bonds with E363, D372.