Lipid-protein interactions as determinants of membrane protein structure and function

Abstract

To determine how the lipid environment affects membrane protein structure and function, strains of Escherichia coli were developed in which normal phospholipid composition can be altered or foreign lipids can be introduced. The properties of LacY (lactose permease) were investigated as a function of lipid environment. Assembly of LacY in membranes lacking PE (phosphatidylethanolamine) results in misorientation of the N-terminal six-TM (transmembrane domain) helical bundle with loss of energy-dependent uphill transport and retention of energy-independent downhill transport. Post-assembly introduction of PE results in nearly native orientation of TMs and restoration of uphill transport. Foreign lipids with no net charge can substitute for PE in supporting native LacY topology, but restoration of uphill transport is dependent on native topology and the proper folding of a solvent-exposed domain. Increasing the positive charge density of the cytoplasmically exposed surface of LacY counters TM misorientation in the absence of neutral lipids, demonstrating that charge interactions between these domains and the surface of the membrane bilayer are determinants of TM orientation. Therefore membrane protein organization or reorganization is determined either during initial assembly or post-insertionally through direct interactions between the protein and the lipid environment, which affects the topogenic potency of opposing charged residues as topological signals independent of the translocon.

Figure 1. Synthesis of native and foreign lipids in E. coli

Pathways, genes encoding the enzyme responsible and major phospholipids naturally occurring in E. coli are indicated in red. Pathways, genes encoding the enzyme responsible and the major lipid synthesized by foreign enzymes in E. coli are indicated in green. 1. cdsA, CDP-diacylglycerol synthase; 2. pssA, phosphatidylserine synthase; 3. psd, phosphatidylserine decarboxylase; 4. pgsA, phosphatidylglycerophosphate synthase; 5. pgpABC, phosphatidylglycerophosphate phosphatase (any of three gene products catalyse this step [18]); 6. cls, CL synthase; 7. mdoB, PG:pre-MDO (membrane-derived oligosaccharide) sn-glycerol-1-phosphate transferase; 8. dgk, diacylglycerol kinase; 9. mgs, GlcDAG synthase (Acholeplasma laidlawii); 10. dgs, GlcGlcDAG synthase (Acholeplasma laidlawii); 11. pcs, PC synthase (Legionella pneumophila). Modified from [9] with permission. © 2009 The American Society for Biochemistry and Molecular Biology.

Figure 2. TM organization of secondary transporters as a function of membrane lipid composition

The top of each structure faces the cytoplasm. Blue, red and yellow/orange rectangles denote TMs numbered sequentially by Roman numerals. N- and C-terminal domains are labelled as NT and CT respectively. Extramembrane domains are labelled according to their exposure to the cytoplasm (C) or periplasm (P) in PE-containing cells. (A) Topology organization of LacY as determined in PE-containing (+PE) cells is depicted. The net charge of each extramembrane domain is noted next to the domain name, and the approximate positions of charged residues throughout the protein are indicated by green (negative charge) and red (positive charge) dots. (B) Topology organization of LacY as determined in PE-lacking (–PE) cells is shown. TM VII (red) exposure to the periplasm results in the loss of salt bridges between the two aspartate residues in TM VII with positively charged amino acids in TM X and TM XI. (C) Topology organization of LacY as determined in cells where assembly of LacY initially occurred in the absence of PE followed by the induction of PE synthesis in the absence of new LacY synthesis. (D) Topology organization of PheP (topology of lipid-insensitive region TM V-CT not shown) as determined in PE-containing cells is shown with labelling of domains following that described in (A). (E) Topology organization of PheP as determined in cells lacking PE. Note that the topological organization of GabP is the same as that of PheP with a slightly different distribution of charged residues. Modified from [9] with permission. © 2009 The American Society for Biochemistry and Molecular Biology.