Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Pur and Pur, in transcriptional repression of the smooth muscle -actin gene in vascular cell types. Although Pur and Pur share substantial sequence homology and nucleic acid-binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Pur is the dominant factor in gene regulation. To dissect the molecular basis of Pur repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Pur with the smooth muscle -actin promoter. Of all the various acidic, basic, and aromatic residues studied, mutation of positionally-conserved arginines in the class I or class II repeat modules significantly attenuated Pur repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA-binding and protein-protein interaction assays were conducted with purified recombinant Pur and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57/96E, and R57/96A each exhibited reduced single-stranded DNA binding affinity for an essential promoter element and diminished interaction with corepressor YB-1/MSY1. Structural analyses of the R57/96A and R57/96E double mutants in comparison to the wild type Pur homodimer revealed aberrant self-association into higher order oligomeric complexes which correlated with decreased -helical content and defective DNA- and protein-binding in vitro. These findings point to a previously unrecognized structural role for certain core arginine residues in forming a conformationally stable Pur protein capable of physical interactions necessary for smooth muscle -actin gene repression.