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

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 1.7 Å resolution. The bovine Lact-C2 structure has a ␤-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 ␤-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membraneinteractive 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.Lactadherin is a M r 47,000 glycoprotein that was identified as a component of milk fat globules. Lactadherin has been known as PAS-6/7, indicating the two glycosylation variants (1), bovine-associated mucoprotein, BA-46, P47, and MFG-E8 (2). Lactadherin is secreted into milk by mammary epithelial cells of humans, cows, and mice (3). The protein is also secreted by some other cells, including aortic medial smooth muscle cells (4), the epithelia of the vas deferens (5, 6), stimulated macrophages (7), and stimulated endothelial cells (8).Bovine lactadherin has a domain structure of EGF1-EGF2-C1-C2 (where EGF indicates epidermal growth factor homology domains). Human lactadherin lacks the first epidermal growth factor homology domain but shares 64% amino acid sequence identity with bovine lactadherin at the other three domains. The EGF2 domain displays an Arg-Gly-Asp motif (9) that binds to the ␣ v ␤ 5 and ␣ v ␤ 3 integrins (1, 10 -12). The C domains of lactadherin share homology with each other and with the discoidin family of lectin domains, including the lipidbinding "C" domains of blood coagulation factor VIII and factor V (2) (homology reviewed by Macedo-Ribeiro et al. (13)). Despite similar nomenclature, these C domains do not share sequence homology with the Ca 2ϩ -binding C2 domains of synaptotagmin, protein kinase C, or phospholipase C. In lactadherin, the se...

The Cation-π Box Is a Specific Phosphatidylcholine Membrane Targeting Motif

“…Additionally, binding of S. aureus PI-PLC enzymes to PC micelles was explored by monitoring the 31 P line width of diC 7 PC in the presence of the protein (Fig. 1C).…”

“…Protein-induced line broadening at 242.76 MHz was measured on a Varian VNMRS 600 spectrometer. For other bacterial PI-PLCs with significant PC affinity, the lipid linewidth increases dramatically around the critical micelle concentration (1.5 mM (10)), as protein-lipid micelle complexes form, then decreases as more diC 7 PC is added to reach a limiting line width. Proteins with weakened affinity for PC have very little effect on the diC 7 PC 31 P line width (28).…”

“…Samples were excited at 282 nm, and changes in the fluorescence intensity at the emission maximum, 337 nm, upon the addition of diC 7 PC were expressed as (I Ϫ I 0 )/I 0 , where I 0 is the emission intensity of protein alone and I is the intensity in the presence of diC 7 PC. A small amount of background signal from pure buffer solution or buffer with different concentrations of diC 7 PC was sub-tracted from the control and sample intensities. The dependence of (I Ϫ I 0 )/I 0 on diC 7 PC concentrations reflects protein binding affinity for that short chain lipid (10,28).…”

“…Proteins with weakened affinity for PC have very little effect on the diC 7 PC 31 P line width (28). Line widths at a given diC 7 PC concentration were measured in duplicate samples.…”

MFG-E8 Regulates Angiogenesis in Cutaneous Wound Healing

“…7e11 The C-terminal domains of MFG-E8 can bind to negatively charged and oxidized phospholipids, 12,13 facilitating opsonization of apoptotic cells for uptake by phagocytes. 10,14 This process has been reported to contribute to autoimmunity, mastitis, sepsis, atherosclerosis, and Alzheimer disease.…”