CHANGE IN CONFORMATION OF PLASMA-MEMBRANE PHOSPHOLIPID SCRAMBLASE INDUCED BY OCCUPANCY OF ITS CA2+ BUILDING SITE

Phospholipid (PL) scramblase is a 35.1 kDa plasma membrane protein tha t mediates the accelerated transbilayer migration of plasma membrane P L in activated, injured, or apoptotic cells exposed to elevated intrac ellular Ca2+. We recently identified a conserved segment in the PL scr amblase polypeptide (residues Asp273 to Asp284) that is essential for its PL-mobilizing function and was presumed to contain the Ca2+ bindin g site of the protein (Zhou, Q., Sims, P. J., and Wiedmer, T. (1998) B iochemistry 37, 2356-2360). Whereas the sequence of this peptide segme nt resembles that of known Ca2+-binding loops within EF-hand containin g proteins, it is unusual in being a single such loop in the entire pr otein and in being closely spaced to the predicted transmembrane helix (Ala291-Gly309), To gain insight into how Ca2+ activates the PL-mobil izing function of PL scramblase, we analyzed conformational changes as sociated with occupancy of this putative Ca2+ binding site. In additio n to activation by Ca2+, the PL-mobilizing function of PL scramblase w as found to be activated by other ions, with apparent affinities Tb3+, La3+ much greater than Ca2+ > Mn2+ > Zn2+ > Sr2+ > Ba2+ > Mg2+. Evide nce for coordinate binding of metal ion by the polypeptide was provide d by resonance energy transfer from protein Trp to Tb3+, which was com peted by excess Ca2+. Metal binding to PL scramblase was accompanied b y increased right-angle light scattering and by a prominent change in circular dichroism, suggesting that coordinate binding of the metal io n induces a conformational change that includes self-aggregation of th e polypeptide, Consistent with this interpretation addition of Ca2+ wa s found to protect PL scramblase from proteolysis by trypsin both in d etergent 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, co nfirming that this suspected EF-hand loopike segment of the polypeptid e directly contributes to the Ca2+ binding site.