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,Gary E Gilbert

doi:10.1074/jbc.m705195200

Chenghua Shao, Valerie A Novakovic + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m705195200

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Abstract

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 1.7A resolution. The bovine Lact-C2 structure has a beta-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 beta-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.

Highlights

Lactadherin is a Mr 47,000 glycoprotein that was identified as a component of milk fat globules
The common feature is a ␤-barrel core with three relatively long loops protruding from one end. Both factor V C2 (fV-C2) and fVIII-C2 have 3– 4 water-exposed hydrophobic residues protruding from long loops, leading to a hypothesis that membrane binding is mediated by insertion of these residues into the membrane
We have found that lactadherin C2 domain (Lact-C2) binds to Ptd-L-Ser-containing membranes with high affinity and that the high affinity binding correlates to a crystal structure that is very similar to those of fV-C2 and fVIII-C2

Summary

The abbreviations used are

Ptd-L-Ser, phosphatidyl-L-serine; Lact-C2, lactadherin C2 domain; fV-C2, factor V C2 domain; fVIII-C2, factor VIII C2 domain; SolC-1, Solution 1 on the C surface of Lact-C2; SolD-1, Solution 1 on the D surface; SolD-2, Solution 2 on the D surface; Lact-C1, lactadherin C1 domain; FITC, fluorescein isothiocyanate; PC, phosphatidylcholine. Homology between the lactadherin C domains and those of factor VIII and factor V correlates with the capacity of lactadherin to compete efficiently for membrane binding sites on Ptd-L-Ser-containing membranes. The capacity of lactadherin to interact with a range of Ptd-L-Ser-containing binding sites and to compete efficiently with coagulation proteins is an unusual feature. Sequence homology between lactadherin C2 domain (Lact-C2) and fV-C2/fVIII-C2 (Fig. 1A) predicts that Lact-C2 will adopt a central ␤-barrel motif and have relatively long loops that display potential membrane-interactive amino acids. Both fV-C2 and fVIII-C2 undergo conformational changes that are related to membrane binding. Conformational docking studies, utilizing the Autodock software package, have identified two hypothetical Ptd-L-Ser binding sites that differ from the previously proposed Ptd-L-Ser binding sites on either fVIII-C2 or fV-C2

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DISCUSSION