Structure-function analysis of mouse Pur beta II. Conformation altering mutations disrupt single-stranded DNA and protein interactions crucial to smooth muscle alpha-actin gene repression.
Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Pur alpha and Pur beta, in transcriptional repression of the smooth muscle alpha-actin gene in vascular cell types. Although Pur alpha and Pur beta share substantial sequence homology and nucleic acid binding properties, genomic promoter and cis-element occupancy studies reported herein suggest that Pur beta is the dominant factor in gene regulation. To dissect the molecular basis of Pur beta repressor activity, site-directed mutagenesis was used to map amino acids critical to the physical and functional interaction of Pur beta with the smooth muscle alpha-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 beta repressor activity in transfected vascular smooth muscle cells and fibroblasts. DNA binding and protein-protein interaction assays were conducted with purified recombinant Pur beta 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 Pur beta homodimer revealed aberrant self-association into higher order oligomeric complexes, which correlated with decreased alpha-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 beta protein capable of physical interactions necessary for smooth muscle alpha-actin gene repression.
Knapp
Anna M
AM
Departments of Biochemistry and Medicine, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
Ramsey
Jon E
JE
Wang
Shu-Xia
SX
Strauch
Arthur R
AR
Kelm
Robert J
RJ
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020868:Gene Silencing; D051379:Mice; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D000199:Actins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; C121485:Pura protein, mouse; C108770:Purb protein, mouse; C541744:Ybx1 protein, mouse; D014157:Transcription Factors