Effects of lipid environment on the conformational changes of an ABC importer

Abstract

In order to shuttle substrates across the lipid bilayer, membrane proteins undergo a series of conformation changes that are influenced by protein structure, ligands, and the lipid environment. To test the effect of lipid on conformation change of the ABC transporter MolBC, EPR studies were conducted in lipids and detergents of variable composition. In both a detergent and lipid environment, MolBC underwent the same general conformation changes as detected by site-directed EPR spectroscopy. However, differences in activity and the details of the EPR analysis indicate conformational rigidity that is dependent on the lipid environment. From these observations, we conclude that native-like lipid mixtures provide the transporter with greater activity and conformational flexibility as well as technical advantages such as reconstitution efficiency and protein stability.

Figure 1. Chemical properties of detergents and lipids. (A) n-Decyl-β-D- maltopyranoside is a common non-ionic detergent used to stabilize TM proteins. The large polar head group and relatively short non-polar tail give the detergent a roughly conical shape, which facilitates the formation of detergent micelles. (B) Phosphatidylethanolamine can represent up to 70% of lipid in bacterial membranes;^, its roughly cylindrical shape enables the formation of lipid bilayers. (C) In addition to triglycerides and hopanoids, phospholipids are an important component of lipid bilayers. Phospholipids contain three components attached to a glycerol backbone: two non-polar tails (R, light gray) and one polar head group (X, dark gray). Here we show a selection of head groups including cardiolipin (CA), phosphatidylglycerol (PG), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI); and major tail groups from E. coli: palmitic acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), 9,10-methylene-hexadecanoic acid (cyclopropyl C17:0), and lactobacillic acid (cyclopropyl C19:0). Chemical structures were prepared using ChemDraw (CambridgeSoft Inc.).

Figure 2. CW-EPR spectroscopy of MolBC_N93C+MTSL and I151C+MTSL showing the effect of lipid reconstitution on TMD conformation change. (A) Ribbon and space fill diagram of MolBC with N93 and I151 shown in black spheres. CW-EPR spectra of MolBC_N93C+MTSL (B-D) and I151C+MTSL (E-G) stabilized in E. coli polar lipid: Egg PC (3:1) liposomes or DM detergent micelles (black or gray respectively). Room temperature spectra (250 Gauss scan width) were recorded apo (B and E), ATP-bound (C and F), and post-hydrolysis (D and G). N93C spectra were normalized by the height of the central peak. I151C spectra were normalized for equal spin (normalized double integration values). MolBC surface and ribbon diagrams were prepared using PyMOL. Spectra were graphed using Grapher 9 (Golden Software Inc.).

Figure 3. Effect of lipid identity on nucleotide-dependent conformation change, shown by CW-EPR. (A) MolB_N93C and MolBC_I151C were spin-labeled with MTSL then reconstituted into soybean PC liposomes (red), E. coli polar lipid: Egg PC (3:1) liposomes (black), or E. coli total lipid liposomes (blue). CW-EPR spectra (250G) were recorded apo, ATP-bound, and post-hydrolysis. For N93C, spectra from samples in E. coli polar: Egg PC are overlaid with soybean PC (A) or E. coli total lipid spectra (B) and were normalized by the height of the central peak. (C) For I151C, the E. coli polar: Egg PC spectra are overlaid with soybean PC and were normalized for equal spin (normalized double integration values).

Figure 4. Schematic of MolBC in a detergent micelle and lipid bilayer. Ribbon and space fill diagram of MolBC with translocation pathway helices 5 and 5a colored cyan on one monomer and magenta on the other. (A) Detergent will form a band of hydrophobic tails that cover the hydrophobic region of MolB and stabilize the protein. When we analyze conformation change at the cytoplasmic region of the transporter, we see that the cytoplasmic gate closes in response to nucleotide binding (red arrows). (B) In the presence of lipid, the binding of ATP causes the NBDs and cytoplasmic gate to close. However, the lipid bilayer appears to resist conformation change of MolBC and allows the transporter less conformational flexibility.