Recognition of negative charge arrays by pleckstrin homology (PH) domains

Pleckstrin homology (PH) domains are typically assumed to be phosphoinositide-binding modules, although most lack strong lipid specificity and their broader ligand repertoire remains poorly defined. We find that many yeast PH domains bind Nsr1p, the ortholog of nucleolin - also identified as a PH domain ligand - suggesting widespread recognition of negatively charged protein regions. The phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5) P ₂ )-binding PLCδ ₁ PH domain also binds a highly phosphorylated region of IRBIT through the same site that recognizes PtdIns(4,5) P ₂ . Using high-throughput integrated phosphopeptide (Hi-P) screening, we surveyed 38,624 mammalian sequences containing three documented phosphoserines. PLCδ ₁ -PH bound numerous phosphorylated and unphosphorylated acidic peptides, and phosphorylation generally strengthened pre-existing interactions rather than conferring strict specificity - without requiring a fixed phosphoserine spacing. Instead, favored phosphopeptides combined a key phosphoserine with upstream acidic residues. Structural modeling suggests that this phosphoserine occupies the canonical inositol phosphate binding pocket, while adjacent acidic residues make delocalized electrostatic contacts. Thus, PH domains can recognize various patterns of protein negative charge, expanding their potential regulatory roles beyond membrane targeting.