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.
Stout
J G
JG
The Blood Research Institute of The Blood Center of Southeastern Wisconsin, Milwaukee 53201-2178, USA.
Zhou
Q
Q
Wiedmer
T
T
Sims
P J
PJ
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008051:Lipid Bilayers; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D013725:Terbium; D002118:Calcium