Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B

Abstract

Purine-rich element binding protein B (Purβ) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purβ with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purβ. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purβ point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purβ. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purβ for ssDNA and diminished the interaction of Purβ with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purβ to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purβ.