Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in annexin B12.
Annexins are a family of soluble proteins that can undergo reversible Ca(2+)-dependent interaction with the interfacial region of phospholipid membranes. The helical hairpins on the convex face of the crystal structure of soluble annexins are proposed to mediate binding to membranes, but the mechanism is not defined. For this study, we used a site-directed spin labeling (SDSL) experimental approach to investigate Ca(2+) and membrane-induced structural and dynamic changes that occurred in the helical hairpins encompassing three of the four D and E helices of annexin B12. Electron paramagnetic resonance (EPR) parameters were analyzed for the soluble and Ca(2+)-dependent membrane-bound states of the following nitroxide scans of annexin B12: a continuous 24-residue scan of the D and E helices in the third repeat (residues 219-242) and short scans encompassing the D-E loop regions of the first repeat (residues 68-74) and the fourth repeat (300-305). EPR mobility and accessibility parameters of most sites were similar when the protein was in solution or in the membrane-bound state, and both sets of data were consistent with the crystal structure of the protein. However, membrane-induced changes in mobility and accessibility were observed in all three loop regions, with the most dramatic changes noted at sites corresponding to the highly conserved serine and glycine residues in the loops. EPR accessibility parameters clearly established that nitroxide side chains placed at these sites made direct contact with the bilayer. EPR mobility parameters showed that these sites were very mobile in solution, but immobilized on the EPR time scale in the membrane-bound state. Since the headgroup regions of bilayer phospholipids are relatively mobile in the absence of annexins, Ca(2+)-dependent binding of annexin B12 appears to form a complex in which the mobility of the D-E loop region of the protein and the headgroup region of the phospholipid are highly constrained. Possible biological consequences of annexin-induced restriction of membrane mobility are discussed.
Isas
Jose Mario
JM
Department of Biochemistry and Molecular Biology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, USA.
Kim
Yujin E
YE
Jao
Christine C
CC
Hegde
Prabhavati B
PB
Haigler
Harry T
HT
Langen
Ralf
R
D000595:Amino Acid Sequence; D017302:Annexins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels
D017302:Annexins; D011994:Recombinant Proteins; D013113:Spin Labels; D002118:Calcium