Structure and dynamics of a helical hairpin that mediates calcium-dependent membrane binding of annexin B12.
A wealth of high-resolution structural data has accumulated for soluble annexins, but only limited information is available for the biologically important membrane-bound proteins. To investigate the structural and dynamic changes that occur upon membrane binding, we analyzed the electron paramagnetic resonance (EPR) mobility and accessibility parameters of a continuous 30-residue nitroxide scan encompassing helices D and E in repeat 2 of annexin B12 (residues 134-163) while the protein was bound to phospholipid vesicles in the presence of Ca(2+). A comparison of these data to those from a previously published study of the protein in solution (Isas, J. M., Langen, R., Haigler, H. T., and Hubbell, W. L. (2002) Biochemistry 41, 1464-1473) showed that the overall backbone fold for the scanned region did not change upon membrane binding. However, side-chains in the loop between the D and E helices were highly dynamic in solution but became essentially frozen in the EPR time scale upon binding to membranes. Accessibility measurements clearly established that side-chains in this loop were exposed to the hydrophobic core of the bilayer and provide the first evidence that a D-E loop directly participates in the Ca(2+)-dependent binding of annexins to membranes. Other localized changes showed that the D-helix became much less dynamic after membrane binding and identified quaternary contact sites in the membrane-bound homo-trimer. Finally, immobilization of the D-E loop upon contact with phospholipid suggests that the bilayer, which is normally very mobile on the EPR time scale, is immobilized in the head-group region by the annexin B12. This suggests that annexin B12 alters membrane structure in a manner that may be biologically significant.
Isas
J Mario
JM
Department of Physiology and Biophysics, University of California, Irvine, 92697, USA.
Langen
Ralf
R
Hubbell
Wayne L
WL
Haigler
Harry T
HT
D017302:Annexins / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D008051:Lipid Bilayers; D008956:Models, Chemical; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D017302:Annexins; D008051:Lipid Bilayers; D010743:Phospholipids; D009569:Nitric Oxide; D010100:Oxygen; D002118:Calcium