Annexin B12 Trimer Formation is Governed by a Network of Protein-Protein and Protein-Lipid Interactions

Membrane protein oligomerization mediates a wide range of biological events including signal transduction, viral infection and membrane curvature induction. However, the relative contributions of protein-protein and protein-membrane interactions to protein oligomerization remain poorly understood. Here, we used the Ca²⁺-dependent membrane-binding protein ANXB12 as a model system to determine the relative contributions of protein-protein and protein-membrane interactions toward trimer formation. Using an EPR-based detection method, we find that some protein-protein interactions are essential for trimer formation. Surprisingly, these interactions are largely hydrophobic, and they do not include the previously identified salt bridges, which are less important. Interfering with membrane interaction by mutating selected Ca²⁺-ligands or by introducing Lys residues in the membrane-binding loops had variable, strongly position-dependent effects on trimer formation. The strongest effect was observed for the E226Q/E105Q mutant, which almost fully abolished trimer formation without preventing membrane interaction. These results indicate that lipids engage in specific, trimer-stabilizing interactions that go beyond simply providing a concentration-enhancing surface. The finding that protein-membrane interactions are just as important as protein-protein interactions in ANXB12 trimer formation raises the possibility that the formation of specific lipid contacts could be a more widely used driving force for membrane-mediated oligomerization of proteins in general.