Crystal Structure of the Bovine Lactadherin C2 Domain, a Potential Anticoagulant, Shows Similarity to Factor V and Factor VIII.
Authors: Lin Lin , Huai Qing , Huang Mingdong , Furie Bruce , Furie Barbara C. 
Published: October 2019

Lactadherin is a glycoprotein expressed by mammary epithelial cells as a cell surface protein and as an abundantly secreted protein during lactation. Lactadherin is comprised of two EGF-like domains and two C-like domains that share homology with the C domains of blood clotting proteins Factor V (FV) and Factor VIII (FVIII). Similar to these coagulation factors, lactadherin binds to phosphatidylserine (PS) containing phospholipid (PL) membranes with high affinity although lactadherin binding requires lower concentrations of PS and is independent of phosphatidylethanolamine. Lactadherin shows efficient competition for membrane binding sites recognized by vitamin K-dependent coagulation factors as well as FV and FVIII with half-maximal displacement of these proteins from PS containing PL vesicles at lactadherin concentrations of 1–4 nM. Lactadherin inhibits the tenase complex, the prothrombinase complex, and the factor VIIa-tissue factor complex. Thus, it has been suggested as an anticoagulant through competition for PL binding sites with blood coagulation proteins. As blood coagulation factors VIII and V bind to PL membranes via C domains with high affinity and specificity, we have determined the crystal structure of the C2 domain of bovine lactadherin (residues 1 to 158) for comparison with the crystal structures of the C2 domains of FV and FVIII. The lactadherin C2 domain was cloned from a bovine EST clone, over-expressed in Pichia pastoris, purified by ion-exchange and gel filtration chromatography and crystallized by the vapor diffusion method. The crystals diffracted to 2.4 Å and have cell dimension of a=108.12Å, b=107.79Å, c= 82.75Å and belong to space group P212121. The structure was determined and refined at a resolution of 2.4 Å. There are four molecules per asymmetry unit. The overall structure of the lactadherin C2 is similar to the C2 domains of FV and FVIII (root-mean-square-deviation of Cα atoms of 0.9 Å and 1.2 Å, respectively). Similarly to the FV (Macedo-Ribeiro, S et al, Nature 1999, 402:434–439) and FVIII (Pratt, KP et al, Nature, 1999, 402:439–441) C2 domains, the lactadherin C2 domain consists of eight major antiparallel strands arranged in two β-sheets of five and three strands packed against one another with the N- and C-terminal regions linked by a disulfide bridge. Like the FV and FVIII C2 domains, the lactadherin C2 domain structure reveals a β-sheet-sandwiched core, from which two β-turns and a loop display a group of solvent-exposed hydrophobic residues. The crystal structures suggest that PL binding is mediated by hydrophobic residues in the C2 domains of FV and FVIII and multiple mutagenesis and antibody inhibition studies support this hypothesis. Based on the crystal structure it is likely that residues Trp26, Phe31, Thr53, Phe81, and Gly82 of lactadherin participate in PL binding. The conformations of regions involved in PL binding are quite different from that of FV and FVIII. The C2 domain of lactadherin may thus have the potential to serve as a unique anticoagulant for treatment of thrombotic disease by blocking the action of FV and FVIII.