Annexin B12 Is a Sensor of Membrane Curvature and Undergoes Major Curvature-dependent Structural Changes

Torsten Fischer,Lucy Lu,Harry T Haigler,Ralf Langen

doi:10.1074/jbc.m611180200

Abstract

The regulation of membrane curvature plays an important role in many membrane trafficking and fusion events. Recent studies have begun to identify some of the proteins involved in controlling and sensing the curvature of cellular membranes. A mechanistic understanding of these processes is limited, however, as structural information for the membrane-bound forms of these proteins is scarce. Here, we employed a combination of biochemical and biophysical approaches to study the interaction of annexin B12 with membranes of different curvatures. We observed selective and Ca(2+)-independent binding of annexin B12 to negatively charged vesicles that were either highly curved or that contained lipids with negative intrinsic curvature. This novel curvature-dependent membrane interaction induced major structural rearrangements in the protein and resulted in a backbone fold that was different from that of the well characterized Ca(2+)-dependent membrane-bound form of annexin B12. Following curvature-dependent membrane interaction, the protein retained a predominantly alpha-helical structure but EPR spectroscopy studies of nitroxide side chains placed at selected sites on annexin B12 showed that the protein underwent inside-out refolding that brought previously buried hydrophobic residues into contact with the membrane. These structural changes were reminiscent of those previously observed following Ca(2+)-independent interaction of annexins with membranes at mildly acidic pH, yet they occurred at neutral pH in the presence of curved membranes. The present data demonstrate that annexin B12 is a sensor of membrane curvature and that membrane curvature can trigger large scale conformational changes. We speculate that membrane curvature could be a physiological signal that induces the previously reported Ca(2+)-independent membrane interaction of annexins in vivo.

Highlights

Control of membrane curvature is an important part of many cellular events [1]
The Effect of Curvature on Membrane Interaction of Annexin B12—To evaluate whether membrane curvature modulates annexin B12 membrane interaction, we developed a gel filtration assay that monitors the binding of annexin B12 to vesicles of different size and lipid composition
In the presence of relatively large (100-nm diameter) phospholipid vesicles (67:33 mol % PS/PC) at pH 7.4, annexin B12 eluted at fractions 24 –28 in the presence of EGTA (Fig. 2A, upper panel) and at fractions 14 –17 in the presence of Ca2ϩ (1 mM) (Fig. 2B, lower panel); this was determined by assaying for the presence of the protein by PAGE followed by Coomassie Blue staining

Summary

Introduction

Control of membrane curvature is an important part of many cellular events [1]. To better understand the molecular mechanisms by which proteins can sense or induce curvature, structural information is essential. It has been suggested that these helical bundles act as a scaffold for membrane curvature [2, 4] While this structural information is clearly important, analysis of the membranebound state is still necessary to fully understand the mechanisms by which proteins interact with curved membranes. The N-terminal region of the BAR domain protein endophilin is known to be important for membrane interaction, yet the crystal structure does not provide any structural information for this region. This region is disordered in the absence of membranes but assumes an amphipathic ␣-helical structure when in contact with membranes [2]. Annexin B12 Is a Sensor of Membrane Curvature of the membrane rather than forming a transmembrane structure [26]

Methods

Results

Conclusion