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J. Biol. Chem., Vol. 282, Issue 19, 14525-14535, May 11, 2007

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Solution NMR Structure of the Barrier-to-Autointegration Factor-Emerin Complex^*

Mengli Cai, Ying Huang, Jeong-Yong Suh, John M. Louis, Rodolfo Ghirlando, Robert Craigie, and G. Marius Clore¹

From the Laboratories of Chemical Physics and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0520

The barrier-to-autointegration factor BAF binds to the LEM domain (Em^LEM) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF₂ dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF₂ and Em^LEM. Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em^LEM bound per BAF₂ dimer. The equilibrium dissociation constant (K_d) for the interaction of the BAF₂ dimer and Em^LEM, determined by isothermal titration calorimetry, is 0.59 ± 0.03 µM. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF₂ dimer in the complex yields a dissociation rate constant of 78 ± 2s^-1. The solution NMR structure of the BAF₂-Em^LEM complex reveals that the LEM and DNA binding sites on BAF₂ are non-overlapping and that both subunits of the BAF₂ dimer contribute approximately equally to the Em^LEM binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF₂ dimer and Em^LEM at the nuclear envelope is discussed.

Received for publication, January 19, 2007 , and in revised form, February 27, 2007.

The atomic coordinates and experimental NMR restraints (accession codes 2ODC for free Em^LEM and 2ODG for the BAF₂-Em^LEM complex) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the Intramural Research Program of the NIDDK, National Institutes of Health and by the AIDS Targeted Antiviral Program of the Office of the Director of the National Institutes of Health (to G. M. C. and R. C.). 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.

¹ To whom correspondence should be addressed: Bldg. 5, NIDKK, National Institutes of Health, Bethesda, MD 20892-0520. Tel.: 301-496-0788; Fax: 301-496-0825; E-mail: mariusc{at}intra.niddk.nih.gov.

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