Abstract
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.
Highlights
A plethora of biological events, including signal transduction[1,2,3,4], viral infection[5,6] and membrane curvature induction[7,8] rely on the ability of peripheral membrane proteins to oligomerize on membranes
The extent of trimer formation was quantified using a previously established, EPR-based method. This method detected trimer formation using spin-spin interaction that occurs as three spin labels at residue 132 (132R1) come into close proximity[36] (Fig. 1b, yellow)
No significant EPR spectral changes were observed when large unilamellar vesicles (LUV) were added to 132R1 in the absence of additional Ca2+ (Fig. 2b), consistent with prior reports that no membrane binding occurs under these conditions[36,42]
Summary
A plethora of biological events, including signal transduction[1,2,3,4], viral infection[5,6] and membrane curvature induction[7,8] rely on the ability of peripheral membrane proteins to oligomerize on membranes. Membranes provide a common interaction surface that increases the local protein concentration and that reduces the dimensionality of protein diffusion from 3D to 2D Both mechanisms have been frequently invoked in order to explain the membrane-mediated aggregation of amyloidogenic proteins[9,15,16,17,18]. We sought to develop an experimental model system that makes it possible to dissect the effects of protein-protein and protein-lipid interaction on the oligomerization of peripheral membrane proteins Toward this end, we used trimer-forming annexin B12 (ANXB12) as a model system. The trimer-forming annexins bind membranes with much higher calcium stoichiometry (~11 Ca2+-ions in the case of ANXB12 as opposed to ~2 in the case of ANXA239). Acronym SB-A SB-E IF-A SBIF-A AB1 AB2 AB3 AB4 AB12 AB13 AB14 AB23 AB24 AB34 AB124 AB2AB′2 AB3AB′2 K1234 AB1K1 AB2K2 AB3K3 AB4K4
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