Journal Article

The role of interfacial lipids in stabilizing membrane protein oligomers

Baldwin, Andrew J., Donlan, Joseph A. C., Hopper, Jonathan T. S., Stansfeld, Phillip J., Struwe, Weston B., Landreh, Michael, Drew, David, Robinson, Carol V., Gupta, Kallol, Uzdavinys, Povilas

2017

Overview

Membrane lipids such as cardiolipin act as molecular glue to preserve the oligomeric states of membrane proteins with low oligomeric stability. Membrane-protein stabilization by lipid binding It is well established that lipid binding leads to the oligomerization of membrane proteins, and hence the activation of many signalling pathways, but it is not clear how the lipid bilayer affects the structure and function of membrane–protein complexes. Carol Robinson and colleagues have developed a mass-spectroscopy-based technique that allows the observation of oligomeric membrane–protein complexes with sufficient resolution to characterize their bound lipids. Using this technique, they evaluate the strength of oligomer formation for some 125 α-helical membrane proteins, including G-protein-coupled receptors. They find that lipid binding modulates protein interfaces, perturbing their monomer–oligomer equilibria, and show that manipulation of lipid binding can modify oligomer stability. And they use modelling to investigate possible binding sites for lipid molecules at the interfaces involved in oligomerization. These findings could aid the optimization of membrane–protein complexes for structural analysis. Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways 1 but is often difficult to define 2 or predict 3 . Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT 4 , one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET 5 , another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na + /H + antiporter NhaA from E. coli , which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/296 / 631/535 / 631/57/2270 / Analysis / Bacterial Proteins - chemistry / Bacterial Proteins - metabolism / Binding Sites - genetics / Biochemistry / biokemi / Cardiolipins - chemistry / Cardiolipins - metabolism / Cardiolipins - pharmacology / Cell Membrane - chemistry / Cell Membrane - metabolism / Cells / Crystal structure / E coli / Escherichia coli - chemistry / Escherichia coli Proteins - chemistry / Escherichia coli Proteins - metabolism / Humanities and Social Sciences / letter / Ligands / Lipids / Lipids - chemistry / Lipids - pharmacology / Mass spectra / Mass spectrometry / Mass Spectrometry - methods / Membrane proteins / Membrane Proteins - chemistry / Membrane Proteins - metabolism / Membranes / Models, Molecular / Molecular Dynamics Simulation / Moritella - chemistry / multidisciplinary / Oligomers / Protein binding / Protein Multimerization - drug effects / Protein Stability - drug effects / Proteins / Receptors, G-Protein-Coupled - chemistry / Receptors, G-Protein-Coupled - metabolism / Science / Signal transduction / Sodium-Hydrogen Exchangers - chemistry / Sodium-Hydrogen Exchangers - metabolism / Thermodynamics / Thermus thermophilus - chemistry