Crystal structure of a self-assembling lipopeptide detergent at 1.20 Å

Abstract

Lipopeptide detergents (LPDs) are a new class of amphiphile designed specifically for the structural study of integral membrane proteins. The LPD monomer consists of a 25-residue peptide with fatty acyl chains linked to side chains located at positions 2 and 24 of the peptide. LPDs are designed to form ␣-helices that selfassemble into cylindrical micelles, providing a more natural interior acyl chain packing environment relative to traditional detergents. We have determined the crystal structure of LPD-12, an LPD coupled to two dodecanoic acids, to a resolution of 1.20 Å. The LPD-12 monomers adopt the target conformation and associate into cylindrical octamers as expected. Pairs of helices are strongly associated as Alacoil-type antiparallel dimers, and four of these dimers interact through much looser contacts into assemblies with approximate D 2 symmetry. The aligned helices form a cylindrical shell with a hydrophilic exterior that protects an interior hydrophobic cavity containing the 16 LPD acyl chains. Over 90% of the methylene/methyl groups from the acylated side chains are visible in the micelle interiors, and Ϸ90% of these adopt trans dihedral angle conformations. Dodecylmaltoside (DDM) was required for the crystallization of LPD-12, and we find 10 -24 ordered DDM molecules associated with each LPD assembly, resulting in an overall micelle molecular weight of Ϸ30 kDa. The structures confirm the major design objectives of the LPD framework, and reveal unexpected features that will be helpful in the engineering additional versions of lipopeptide amphiphiles.de novo protein design ͉ detergent design ͉ membrane proteins ͉ self-assembling amphiphiles ͉ x-ray crystallography D etergents that are able to stabilize native structures of membrane proteins in the absence of lipid bilayers are essential tools for the structural study of membrane proteins (1, 2). Despite the large number of detergents that are available, no single detergent is optimal for all purposes, and choice of the solubilizing agent is highly dependent on both the target protein and on the application. In particular, the structural study of membrane proteins by NMR or x-ray crystallography depends critically on the choice of detergent (3, 4), and many proteins cannot be studied because of problems with protein stability and aggregation in the commonly used detergents (5). Because of these issues, there is a need to expand the range of amphiphiles available for membrane proteins research.Nondenaturing detergents act as membrane mimetics, and an ideal detergent would generate a local environment at the protein surface that is indistinguishable from that of the native lipid bilayer. A central shortcoming of many of the commonly used detergents is that the micelle interior is less ordered and less well-packed relative to the interiors of lipid bilayers (2, 6). This is a direct result of the shape of the amphiphile monomer, which has a high degree of positive intrinsic curvature, resulting in self-assembly into spherical or ellipsoidal micelles with...

New amphiphiles for membrane protein structural biology

“…Like steroid-based facial amphiphiles, these lipopeptides form small, tightly packed and rigid oligomers. Crystal structures of lipopeptides with bound DDM molecules showed a cylindrical bundle of eight helical peptides assembled with the hydrophobic chains buried inside, supporting a model proposed for the sequestration of MPs in lipopeptide micelles [47, 48]. The lipopeptides are relatively costly, making their use impractical except in the final stages of sample preparation via detergent exchange.…”

Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure

“…This follows from the fact that when LPD-12, which has a much smaller micellar size (radius ∼20 Å) than Triton X-100 (radius ∼40 Å), is used for spike solubilization, the bottom appears to exclude the negative stain much less than when Triton X-100 is used (Fig. S2, columns 2, 4, and 5, side 2 view) (28,38,39). The endodomain of gp41 cannot be part of the reconstruction as this has been deleted from the spike analyzed.…”

“…Finally, we considered the possibility that the exchange of the lipid membrane of the VLP with the Triton X-100 micelle(s) during solubilization might induce aberrant features in the spike. To this end we exchanged the Triton X-100 of solubilized spikes, without cross-linking, with a lipopeptide detergent (LPD-12) and determined its 3D structure using negatively stained samples (28). LPDs have been shown to stabilize membrane proteins in solution better than traditional detergents (29).…”

Insight into the Self-Assembling Properties of a Peptergent: A Molecular Dynamics Study

“…When a membrane protein is present, the peptide steadily forms a beta sheet structure at the protein surface and is able to displace DPC surfactants. Tao et al proposed a model for the organization of the peptides around an IMP [9]. The peptides are thought to generate a beta-barrel belt around the hydrophobic helical domain that could help stabilize purified membrane proteins [13].…”