Structure-Function Analysis of Mouse Purβ II: CONFORMATION ALTERING MUTATIONS DISRUPT SINGLE-STRANDED DNA AND PROTEIN INTERACTIONS CRUCIAL TO SMOOTH MUSCLE α-ACTIN GENE REPRESSION

• Published in: The Journal of biological chemistry 2007-12, Vol.282 (49), p.35899-35909
• Main Authors: Knapp, Anna M, Ramsey, Jon E, Wang, Shu-Xia, Strauch, Arthur R, Kelm, Robert J. Jr
• Format: Article
• Language: English
• Subjects: Actins - biosynthesis; Actins - genetics; Amino Acid Substitution; Animals; Cell Line; DNA, Single-Stranded - genetics; DNA, Single-Stranded - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Fibroblasts - cytology; Fibroblasts - metabolism; Gene Silencing; Mice; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - metabolism; Mutagenesis, Site-Directed; Mutation, Missense; Myocytes, Smooth Muscle - cytology; Myocytes, Smooth Muscle - metabolism; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Protein Binding - genetics; Protein Structure, Quaternary; Protein Structure, Secondary; Rats; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Repressor Proteins - genetics; Repressor Proteins - metabolism; Response Elements - physiology; Sequence Homology, Amino Acid; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M706617200

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Purα and Purβ, in transcriptional repression of the smooth muscle α-actin gene in vascular cell types. Although Purα and Purβ share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Purβ is the dominant factor in gene regulation. To dissect the molecular basis of Purβ repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Purβ with the smooth muscle α-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 Purβ repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Purβ and selected mutants to reveal the physical basis for loss-of-function. Mutants R57E, R57E/R96E, and R57A/R96A 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 R57A/R96A and R57E/R96E double mutants in comparison to the wild-type Purβ homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased α-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 Purβ protein capable of physical interactions necessary for smooth muscle α-actin gene repression.