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J. Biol. Chem., Vol. 283, Issue 11, 7230-7241, March 14, 2008

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Crystal Structure of Lactadherin C2 Domain at 1.7Å Resolution with Mutational and Computational Analyses of Its Membrane-binding Motif^*

Chenghua Shao, Valerie A. Novakovic, James F. Head, Barbara A. Seaton, and Gary E. Gilbert¹

From the Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118 and the Department of Medicine, Veterans Affairs Boston Healthcare System, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02132

Lactadherin is a phosphatidyl-L-serine (Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-L-Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain (Lact-C2) at 1.7Å resolution. The bovine Lact-C2 structure has a β-barrel core that is homologous with the factor VIII C2 (fVIII-C2) and factor V C2 (fV-C2) domains. Two loops at the end of the β-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-L-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.

Received for publication, June 25, 2007 , and in revised form, December 18, 2007.

The atomic coordinates and structure factors (code 3BN6) 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 a Veterans Affairs Merit Award (to G. E. G.). 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 Table S1 and Figs. S1-S3.

¹ To whom correspondence should be addressed: Veterans Affairs Boston Healthcare System, 1400 VFW Parkway, West Roxbury, MA 02132. Tel.: 857-203-5252; Fax: 857-203-5592; E-mail: ggilbert{at}rics.bwh.harvard.edu.

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