Change in conformation of plasma membrane phospholipid scramblase induced by occupancy of its Ca2+ binding site

Phospholipid (PL) scramblase is a 35.1 kDa plasma membrane protein that mediates the accelerated transbilayer migration of plasma membrane PL in activated, injured, or apoptotic cells exposed to elevated intracellular Ca2+. We recently identified a conserved segment in the PL scramblase polypeptide (residues Asp273 to Asp284) that is essential for its PL-mobilizing function and was presumed to contain the Ca2+ binding site of the protein (Zhou, Q., Sims, P. J., and Wiedmer, T. (1998) Biochemistry 37, 2356-2360). Whereas the sequence of this peptide segment resembles that of known Ca2+-binding loops within EF-hand containing proteins, it is unusual in being a single such loop in the entire protein and in being closely spaced to the predicted transmembrane helix (Ala291-Gly309). To gain insight into how Ca2+ activates the PL-mobilizing function of PL scramblase, we analyzed conformational changes associated with occupancy of this putative Ca2+ binding site. In addition to activation by Ca2+, the PL-mobilizing function of PL scramblase was found to be activated by other ions, with apparent affinities Tb3+, La3+ >> Ca2+ > Mn2+ > Zn2+ > Sr2+ >> Ba2+, Mg2+. Evidence for coordinate binding of metal ion by the polypeptide was provided by resonance energy transfer from protein Trp to Tb3+, which was competed by excess Ca2+. Metal binding to PL scramblase was accompanied by increased right-angle light scattering and by a prominent change in circular dichroism, suggesting that coordinate binding of the metal ion induces a conformational change that includes self-aggregation of the polypeptide. Consistent with this interpretation, addition of Ca2+ was found to protect PL scramblase from proteolysis by trypsin both in detergent solution as well as in situ, within the erythrocyte membrane. Mutation in the segment Asp273-Asp284 reduced Tb3+ incorporation and attenuated the change in CD spectrum induced by bound metal ligand, confirming that this suspected EF-hand loopike segment of the polypeptide directly contributes to the Ca2+ binding site.