Structure of membrane-bound annexin A5 trimers: A hybrid Cryo-EM - X-ray crystallography study

Annexins constitute a family of phospholipid- and Ca2+-binding proteins inv olved in a variety of membrane-related processes. The property of several a nnexins, including annexin A5, to self-organize at the surface of lipid mem branes into 2D ordered arrays has been proposed to be functionally relevant in cellular contexts. To further address this question, we investigated th e high-resolution structure of annexin A5 trimers in membrane-bound 2D crys tals by cryo-electron microscopy (Cryo-EM). A new 2D crystal form was disco vered, with p32(1) symmetry, which is significantly better ordered than the 2D crystals reported before. A 2D projection map was obtained at 6.5 Angst rom resolution, revealing protein densities within each of the four domains characteristic of annexins. A quantitative comparison was performed betwee n this structure and models generated from the structure of the soluble for m of annexin A5 in pseudo-R3 3D crystals. This analysis indicated that both structures are essentially identical, except for small local changes attri buted to membrane binding. As a consequence, and contrary to the common vie w, annexin A5 molecules maintain their bent shape and do not flatten upon m embrane binding, which implies either that the four putative Ca2+ and membr ane-binding loops present different types of interaction with the membrane surface, or that the membrane surface is locally perturbed. We propose that the trimerization of annexin A5 molecules is the relevant structural chang e occurring upon membrane binding. The evidence that 2D arrays of annexin A 5 trimers are responsible for its in vitro property of blood coagulation in hibition supports tl-Lis conclusion. (C) 2000 Academic Press.