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.