Stabilization and Crystallization of a Membrane Protein Involved in Lipid Transport

Lipoteichoic acids (LTA) are ubiquitous cell wall components of Gram-positive bacteria. In Staphylococcus aureus LTA are composed of a polymer with 1,3-linked glycerol phosphate repeating units anchored to the plasma membrane. The anchor molecule is a lipid-linked disaccharide (anchor-LLD) synthesized at the cytoplasmic leaflet of the membrane. The anchor lipid becomes accessible at the outer leaflet of the membrane after the flippase LtaA catalyzes translocation. Recently we have elucidated the structure of LtaA using vapor diffusion X-ray crystallography and in situ annealing. We were able to obtain LtaA crystals after optimization of purification protocols that led to stabilization of LtaA isolated in detergent micelles. Here we report a protocol that describes the purification, stabilization, crystallization, and data collection strategies carried out to determine the structure of LtaA. We highlight key points that can be used to determine crystal structures of other membrane proteins

Solution Structures of Engineered Vault Particles

Prior crystal structures of the vault have provided clues of its structural variability but are non-conclusive due to crystal packing. Here, we obtained vaults by engineering at the N terminus of rat major vault protein (MVP) an HIV-1 Gag protein segment and determined their near-atomic resolution (∼4.8 Å) structures in a solution/non-crystalline environment. The barrel-shaped vaults in solution adopt two conformations, 1 and 2, both with D39 symmetry. From the N to C termini, each MVP monomer has three regions: body, shoulder, and cap. While conformation 1 is identical to one of the crystal structures, the shoulder in conformation 2 is translocated longitudinally up to 10 Å, resulting in an outward-projected cap. Our structures clarify the structural discrepancies in the body region in the prior crystallography models. The vault's drug-delivery potential is highlighted by the internal disposition and structural flexibility of its Gag-loaded N-terminal extension at the barrel waist of the engineered vault