NMR solution structure of C2 domain of MFG-E8 and insights into its recognition with phosphatidylserine

Abstract

MFG-E8 (also known as lactadherin), which is a secreted glycoprotein from a variety of cell types, possesses two EGF domains and tandem C domains with sequence homology to that of blood coagulation proteins factor V and factor VIII. MFG-E8 binds to phosphatidylserine (PS) in membranes with high affinity. We have recently shown that the C2 domain of MFG-E8 bears more specificity toward PS when compared with phophatidylcholine (PC), another phospholipid thought to be involved in the immune function of phagocytes. In our current study, we have determined the solution structure of the C2 domain by nuclear magnetic resonance (NMR) spectroscopy, characterized the molecular basis of binding between the C2 domain and PS by 31P-NMR spectroscopy. Furthermore, we also verified that positively charged and aromatic residues clustered in loops 1-3 of the C2 domain play key roles in recognizing PS in apoptotic cells. Last but not least, the influence of C1 domain on PS binding was also tested in our new Co-culture FACS analysis and confirmed its role in the binding event.