Tubulation by amphiphysin requires concentration-dependent switching from wedging to scaffolding.

BAR proteins are involved in a variety of membrane remodeling events but how they can mold membranes into different shapes remains poorly understood. Using electron paramagnetic resonance, we find that vesicle binding of the N-BAR protein amphiphysin is predominantly mediated by the shallow insertion of amphipathic N-terminal helices. In contrast, the interaction with tubes involves deeply inserted N-terminal helices together with the concave surface of the BAR domain, which acts as a scaffold. Combined with the observed concentration dependence of tubulation and BAR domain scaffolding, the data indicate that initial membrane deformations and vesicle binding are mediated by insertion of amphipathic helical wedges, while tubulation requires high protein densities at which oligomeric BAR domain scaffolds form. In addition, we identify a pocket of residues on the concave surface of the BAR domain that insert deeply into tube membrane. Interestingly, this pocket harbors a number of disease mutants in the homologous amphiphysin 2.

Computer modeling of nitroxide spin labels on proteins.

Electron paramagnetic resonance using site-directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances. We describe an algorithm, PRONOX, for rapid computation of interlabel distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12 and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein, a total of 44 interlabel distances. The program reproduced these distances accurately (r(2) = 0.94, slope = 0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggest that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure.

Roles of amphipathic helices and the bin/amphiphysin/rvs (BAR) domain of endophilin in membrane curvature generation.

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions. Spin-labeled side chains on the concave surface of the BAR domain do not penetrate into the acyl chain interior, indicating that the BAR domain interacts only peripherally with the surface of a curved bilayer. Using a combination of EPR data and computational refinement, we determined the structure of residues 63-86, a region that is disordered in the crystal structure of rat endophilin A1. Upon membrane binding, residues 63-75 in each subunit of the endophilin dimer form a slightly tilted, amphipathic alpha-helix that directly interacts with the membrane. In their predominant conformation, these helices are located orthogonal to the long axis of the BAR domain. In this conformation, the amphipathic helices are positioned to act as molecular wedges that induce membrane curvature along the concave surface of the BAR domain.

Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in annexin B12.

Annexins are a family of soluble proteins that can undergo reversible Ca(2+)-dependent interaction with the interfacial region of phospholipid membranes. The helical hairpins on the convex face of the crystal structure of soluble annexins are proposed to mediate binding to membranes, but the mechanism is not defined. For this study, we used a site-directed spin labeling (SDSL) experimental approach to investigate Ca(2+) and membrane-induced structural and dynamic changes that occurred in the helical hairpins encompassing three of the four D and E helices of annexin B12. Electron paramagnetic resonance (EPR) parameters were analyzed for the soluble and Ca(2+)-dependent membrane-bound states of the following nitroxide scans of annexin B12: a continuous 24-residue scan of the D and E helices in the third repeat (residues 219-242) and short scans encompassing the D-E loop regions of the first repeat (residues 68-74) and the fourth repeat (300-305). EPR mobility and accessibility parameters of most sites were similar when the protein was in solution or in the membrane-bound state, and both sets of data were consistent with the crystal structure of the protein. However, membrane-induced changes in mobility and accessibility were observed in all three loop regions, with the most dramatic changes noted at sites corresponding to the highly conserved serine and glycine residues in the loops. EPR accessibility parameters clearly established that nitroxide side chains placed at these sites made direct contact with the bilayer. EPR mobility parameters showed that these sites were very mobile in solution, but immobilized on the EPR time scale in the membrane-bound state. Since the headgroup regions of bilayer phospholipids are relatively mobile in the absence of annexins, Ca(2+)-dependent binding of annexin B12 appears to form a complex in which the mobility of the D-E loop region of the protein and the headgroup region of the phospholipid are highly constrained. Possible biological consequences of annexin-induced restriction of membrane mobility are discussed.