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J. Biol. Chem., Vol. 282, Issue 49, 35899-35909, December 7, 2007

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Structure-Function Analysis of Mouse Purβ II

CONFORMATION ALTERING MUTATIONS DISRUPT SINGLE-STRANDED DNA AND PROTEIN INTERACTIONS CRUCIAL TO SMOOTH MUSCLE -ACTIN GENE REPRESSION^*

Anna M. Knapp¹, Jon E. Ramsey², Shu-Xia Wang, Arthur R. Strauch^¶, and Robert J. Kelm, Jr.³

From the Departments of Biochemistry and Medicine, Cardiovascular Research Institute, University of Vermont College of Medicine, Burlington, Vermont 05405 and the ^¶Department of Physiology and Cell Biology, Dorothy M. Davis Heart & Lung Research Institute, Ohio State University College of Medicine and Public Health, Columbus, Ohio 43210

Previous studies from our laboratories have implicated two members of the Pur family of single-stranded DNA/RNA-binding proteins, Pur and Purβ, in transcriptional repression of the smooth muscle -actin gene in vascular cell types. Although Pur 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 -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 -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 -actin gene repression.

Received for publication, August 9, 2007 , and in revised form, September 28, 2007.

^* This study was funded in part by Grant HL054281 (to R. J. K.) from the National Heart, Lung, and Blood Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5 and Table S1.

¹ Supported by NHLBI, National Institutes of Health Institutional Training Grant T32 HL007594.

² Supported by an American Heart Association Predoctoral Fellowship 0515620T.

³ To whom correspondence should be addressed: Colchester Research Facility, 208 South Park Dr., Colchester, VT 05446. Tel.: 802-656-0329; Fax: 802-656-8969; E-mail: Robert.Kelm{at}uvm.edu.

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