Substrate Specificity of the Aminophospholipid Flippase and Other Phosphatidylserine Binding Proteins

Abstract

The generation and maintenance of the asymmetric distribution of phosphatidylserine across the cell plasma membrane is regulated by an ATP-dependent, substrate specific lipid flippase. Transport activity is strongly dependent on the structure of its preferred substrate, 1,2-sn-glycerophospho-L-serine. With the exception of methylation of the primary amine group, any modification to the structural elements comprising the polar portion of the molecule results in significantly reduced rates of substrate transport. Substrate lipids are stereospecifically recognized by the flippase. The 1,2-sn-glycerol isomer, but not the 2,3-sn-glycerol isomer of PS is transport competent, but the enzyme does not distinguish between the L- and D-serine isomers of PS. This substrate specificity defines the characteristics of the lipid binding site and also provides biochemical criteria for the identification of putative flippases. By searching for ATPases that are stereoselectively activated by PS, a candidate flippase has been purified from human erythrocytes. This enzyme is uniquely activated by PS in both detergent micelles and in phospholipid bilayers, and demonstrates a stereochemical specificity identical to that expressed by the flippase. A member of a new class of P-type ATPases, which have been associated with PS flippase activity, has been expressed in insect cells and purified. This enzyme (ATP8A1) is also activated exclusively by the 1,2-sn-glycerol isomer of PS, regardless of the stereochemistry of the serine headgroup. The substrate specificity expressed by the flippase and these two ATPases is unique among PS-binding proteins, including blood clotting factors, protein kinase C or the macrophage PS receptor, each of which interact only with the L-serine, and not the D-serine isomer of PS. These studies indicate that at least two distinct PS binding motifs have evolved to enable proteins to selectively interact with PS.