Modulation by Ca²⁺ and by Membrane Binding of the Dynamics of Domain III of Annexin 2 (p36) and the Annexin 2−p11 Complex (p90):  Implications for Their Biochemical Properties

Abstract

The modulation of the local structure and dynamics of domain III of annexin 2 (Anx2), in both the monomeric (p36) and heterotetrameric forms (p90), by calcium and by membrane binding was studied by time-resolved fluorescence intensity and anisotropy measurements of the single tryptophan residue (W212). The results yield the same dominant excited-state lifetime (1.4 ns) in both p36 and p90, suggesting that the conformation and environment of W212 are very similar. The fluorescence anisotropy decay data were analyzed by associative (two-dimensional) as well as nonassociative (one-dimensional) models. Although no statistical criterion is decisive for one model versus the other, only the associative model allows recovery of a physically relevant value of the Brownian rotational correlation of the protein. Using the associative model, a nanosecond flexibility is detectable in p90 but not in p36. When Ca(2+) binds in the millimolar concentration range to both forms of Anx2, a conformational change takes place leading to an increase of the major excited-state lifetime (2.6 ns) and to a suppression of the W212 local flexibility of p90. Binding to membranes of either p36 or p90 in the presence of Ca(2+) does not induce any conformational change other than that provoked by Ca(2+) binding alone. The W212 local flexibility in both proteins increases significantly, however, in their membrane-bound forms. In the presence of membranes, the conformation change of domain III in p90 displays a sensitivity to Ca(2+) 2 orders of magnitude higher than that of p36, reaching intracellular sub-micromolar concentration ranges. This higher Ca(2+) sensitivity correlates with the Ca(2+)-dependent membrane aggregation but not with their Ca(2+)-dependent binding to membranes. The significance of these structural and dynamical changes for the function of the protein is discussed.

Identification of Annexin II Heterotetramer as a Plasmin Reductase

“…The cells were subsequently sonicated in lysis buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 10 mM MgCl 2 , 2 mM dithiothreitol ϩ protease inhibitors) and centrifuged at 100,000 ϫ g for 1 h at 4°C. Both p11 mutants were purified in the same manner as wild-type p11 (23). Briefly, the cell lysis supernatant was precipitated with 50% (NH 4 ) 2 SO 4 , and the supernatant was applied to a butylSepharose column equilibrated in lysis buffer containing 50% (NH 4 ) 2 SO 4 .…”

Novel Organization and Properties of Annexin 2-Membrane Complexes

“…In the case of AnxA5, it results in the swinging out of Trp-187 in repeat III from a buried to a solvent-exposed position (10 -14). Trp-212 in AnxA2 detects also a Ca 2ϩ -induced conformational change in repeat III, but this residue remains buried in the protein (15,16). The major difference between the membrane-free and the membrane-bound forms of annexin 2 is the Ca 2ϩ sensitivity.…”

The N-terminal Domain of Annexin 2 Serves as a Secondary Binding Site during Membrane Bridging

“…Others, including AnxA1 and AnxA2 form membrane junctions with or without protein ligands attached to their N-terminal segment (7)(8)(9)(10)(11). The classical mechanism described for these two processes involves a primary Ca 2ϩ -dependent membrane binding step leading to conformational changes of the core domain, in particular of repeat III (12)(13)(14)(15)(16)(17)(18). The N-terminal domain of Anxs also plays an important role in the specific membrane aggregative properties.…”

“…The N-terminal domain of Anxs also plays an important role in the specific membrane aggregative properties. In particular, the N-terminal domain of AnxA1 and AnxA2 interacts strongly with proteins of the S100 family, resulting in a severalfold increase in the Ca 2ϩ sensitivity of membrane aggregation (17). Recently, we showed that the N-terminal domain of monomeric AnxA2 was also involved in the Ca 2ϩ -dependent membrane bridging at neutral pH: we demonstrated interactions between the N-terminal domains of two adjacent AnxA2 molecules, which were not in direct contact with the membrane phospholipids (19).…”