The role of interfacial lipids in stabilizing membrane protein oligomers

Abstract

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. 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, 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, 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.

Extended Data Figure 1. Mass spectra of LeuT recorded with increasing collision voltages and of a LeuT fusion protein construct.

a, Mass spectra of LeuT, liberated from OG micelles, (green/grey spheres, most abundant charge state highlighted in pale blue), show that the 7.4 kDa lipid adduct (blue/purple head groups) is retained throughout the trap collision energy range (white, blue arrow) of the mass spectrometer. b, Mass spectra of LeuT expressed as a fusion protein with eYFP (LeuT-eYFP yellow circles), liberated from OG micelles, show that the dimer is similarly associated with a 7.4 kDa adduct.

Extended Data Figure 2. Mass spectra of LeuT following incubation with delipidating detergents and E. coli polar lipids.

a, Mass spectrum of LeuT liberated from OG micelles (green head groups) shows low-abundance, delipidated monomers (green spheres, 59.3 kDa) and high-abundance, lipid-bound dimers (green/black spheres, 126.0 kDa). b, Mass spectrum of LeuT after incubation with neopentyl glycol (NG, orange head-groups) shows only delipidated monomers. c, Mass spectrum of LeuT in OG, after incubation with NG, shows only delipidated monomers. d, Mass spectrum recorded after incubation of delipidated LeuT monomers, in OG, with E. coli polar lipids (blue/purple head-groups) shows delipidated monomers and lipid-bound dimers. e, Mass spectrum recorded after adding dilysocardiolipin (blue head-groups) to delipidated monomeric LeuT in OG (c) shows no dimerisation in the presence of this lipid.

Extended Figure 3. High energy MS/MS experiment of the 23+ charge state of dimeric LeuT, with the 7.4 kDa adduct, as a function of collision voltage.

Three satellite peaks represent the lipid bound states arising through the dissociation of the monomer. The naked monomer is highlighted in blue, while the three satellite peaks assigned to one phospholipid (PL), one CDL and three PL bound species (red, green and yellow respectively). Under higher energy, only the CDL bound species remains, discounting the mathematical possibility of two PL bound species. Inset shows the isolated 23+charge state of the lipid bound dimer. Presence of bound CDL at higher energy, over PL indicates a higher binding energy of CDL over the latter, plausibly owing to greater ionic and hydrophobic interactions.

Extended Data Figure 4. Site directed mutagenesis of selected residues at the LeuT dimer interface, resulting mass spectra and MD simulations.

a, Mass spectrum of LeuT F488AY489A, liberated from OG micelles, reveals monomeric LeuT (green spheres). Inset shows the LeuT dimer interface, with key π-stacking interactions (yellow dotted lines, distances labelled in red) and between aromatic residues (purple). When residues F488 and Y489 (orange arrows) are mutated to alanine the π-stacking interactions are abolished and LeuT cannot dimerize. b, MD simulations of LeuT in an E. coli lipid bilayer reveal possible binding sites of interfacial phospholipids and CDL (upper panel, viewed from cytoplasmic side of membrane). The CDL phosphate groups (orange) interact closely with positively charged residues (K376, H377, R506, blue) at the dimer interface. Phosphoethanolamine (PE) and phosphatidylglycerol (PG) also bind at the dimer interface. c, Mass spectrum of LeuT expressed in a CDL-deficient E. coli strain (BKT22), liberated from OG micelles, shows monomeric LeuT, implying that CDL is required for LeuT dimerisation. d, Mass spectrum of LeuT K376AH377A, liberated from OG micelles, shows monomeric LeuT.

Extended Data Figure 5. Coarse-grained MD simulations on LeuT and NhaA dimer.

Particle densities from five repeats of 1 μs Coarse-grained MD simulations for CDL around (a) LeuT. The surface densities represent the most occupied positions from the simulations of the phosphate (orange), glycerol (red) and alkyl tails (purple) particles of CDL. As the figures show the proposed binding sites at the interface are the only places where CDL shows considerable population density. Comparative particle densities of (b) CDL, (c) PG and (d)PE at the LeuT dimeric interface, summed over the simulations show no/minimal densities of PG and PE at the CDL binding site. (a)-(d) together shows that the proposed binding sites of CDL at the interface are sites of specific bindings. e, Dimeric structure of LeuT with modelled APT (aminopentanetetrol, aminophospholipids) classes of lipid present in the A. Aeolicus . The lipid was drawn in ChemDraw and subsequently modelled by superimposing it on the CDL to the CDL bound dimeric structure. The favorable van der Waals’ distances show that it is capable of bridging the dimeric entity, through the same sets of residues that were found to be critical towards CDL binding, in an endogenous environment lacking CDLs. f, Particle densities from five repeats of 1 μs Coarse-grained MD simulations for CDL (phosphate group in orange, glycerol in red and alkyl tails in purple) and POPG (in blue) around NhaA dimer interface. As before, the density of CDL is considerably higher than that of PG. Although Unlike LeuT, here the difference between the density of CDL and PG is lower, suggesting this site has lesser exclusivity towards CDL than that in LeuT. Indeed, MS analysis shows a heterogenous distribution of lipids with dimeric NhaA, with mostly CDL but some amount bound phospholipids.

Extended Data Figure 6. Mass spectra of His-tagged and unmodified SemiSWEET and identification of endogenous and exogenous lipid binding

a, Mass spectrum of unmodified SemiSWEET, liberated from tetraethyleneglycolmonooctyl ether (C8E4) micelles, reveals SemiSWEET monomers and dimers (black spheres). b, Mass spectrum of deca-His tagged SemiSWEET, liberated from C8E4 micelles, reveals SemiSWEET monomers and dimers (green spheres). c, High energy MS/MS of unmodified SemiSWEET, liberated from dodecylmaltoside (DDM) micelles, allows isolation of the 6+ charge state (black spheres) of the SemiSWEET monomer (black spheres) bound to endogenous lipids. Fragmentation of the lipid-bound species leads to loss of either cardiolipin (1470 ± 26 Da, purple head-groups), 1 or 2 neutral phospholipids (each 756 ± 22 Da, blue head-groups), or a positively charged phospholipid. Trap collision voltages shown in white inside blue arrow. d, Mass spectrum of deca-His SemiSWEET, liberated from C8E4 micelles and incubated with phosphatidylglycerol (PG, blue head-groups). PG binds to both monomers and dimers (dotted boxes highlight lipid-bound peaks) without substantial preference. e, Mass spectrum recorded after incubation in solution of an equimolar ratio of deca-His tagged and untagged SemiSWEET (green and black spheres, respectively), liberated from tetraethyleneglycolmonooctyl ether (C₈E₄) micelles. Plot of the percentage abundance of hetero- and homo- dimers over time (inset) SemiSWEET heterodimers (red trace, peaks highlighted red in mass spectrum) and homodimers (black trace), revealing the solution phase monomer-dimer equilibrium (inset, PDB ID 4QND).

Extended Data Figure 7. Mass spectrum and high-energy MS/MS of NhaA at a range of collision voltages

a, Mass spectrum of NhaA, liberated from C8E4 micelles, reveals NhaA monomers (green spheres) bound to CDL (purple head-groups) and an ensemble of NhaA dimer species in different lipidation states (highlighted in green). b, MS/MS of the 15+ charge state (green) of the NhaA dimer (green/black spheres) bound to 2 CDL liberated from C8E4 micelles. Increasing collision voltage applied to the 2 x CDL-bound species leads to: loss of 1 CDL to form NhaA dimers bound to 1 CDL (40 V); loss of 2 CDL to form delipidated NhaA dimers, with concomitant generation of NhaA monomers (70 V) and further dissociation of NhaA dimers into monomers (120 V). Trap collision voltages (white inside blue arrow).

Extended Data Figure 8. Sequence and structure alignment of LeuT with other eukaryotic biogenic transporters

a, The basic residues of LeuT that are involved in lipid binding (red box) are conserved across the BATs. b, Two views of the superimposed structures of LeuT (PDB ID 2A65, black) and SERT (PDB ID 5I6Z, light blue) show the differences in the dimer interface. Dimer interface helices are highlighted with arrows and coloured (LeuT green, SERT red); basic residues responsible for lipid binding in LeuT (yellow mesh). One of the interface helices in SERT swings away from the interface, negating the possibility of lipid-induced oligomerisation analogous to that proposed for LeuT.

Figure 1. Plot of buried surface area and number of salt bridges for oligomeric α-helical membrane proteins and native mass spectra.

Protein oligomers are represented by circles color coded according to the number of salt bridges and are grouped by oligomeric state (pentamers+ oligomeric state ≥ 5). A random horizontal jitter has been applied to all points to aid visualisation. NhaA and LeuT (outlined in red) are two of the weakest oligomers having one of the lowest buried surface areas and no salt bridges. 12 proteins for which mass spectra have been recorded, are outlined in green. Illustrated are mass spectra of trimeric AmtB, tetrameric AqpZ and pentameric ELIC. A larger buried surface area than LeuT and NhaA, but absence of salt bridges, make SemiSWEET a relatively stronger dimer than LeuT and NhaA but weaker than the other 12 oligomeric proteins.

Figure 2. Schematic of the high-energy tandem MS (HE-MS) platform, mass spectra and molecular dynamics (MD) simulations of the lipid-bound LeuT dimer.

a, Protein-lipid complexes (red/green rods, yellow) are liberated from detergent micelles (blue) by a high potential difference applied to the cone (box 1). Quadrupole isolation of the protein-lipid complex separates it from detergent molecules and bulk lipids (box 2). Collision with neutral gas in the collision cell dissociates the protein-lipid complex (box 3). Masses of constituent protein and lipids are measured in the Time-of-Flight analyser. b, Mass spectrum of LeuT liberated from OG micelles shows monomers (green) and dimers (green/grey) with a mass 7.4 kDa greater than the amino acid sequence mass. c, MS/MS of 23+ charge state of LeuT reveals monomers with cardiolipin (CDL, purple head-group) and phospholipid (PL, blue head-group) retained. Masses of the bound lipids are marked black (PL) and red (CDL). Inset: MD simulation of LeuT in an E. coli lipid bilayer revealing possible binding sites of interfacial CDL. The CDL phosphate groups (orange) interact closely with positively charged residues (K376, H377, R506, blue) at the dimer interface. Interactions are shown (yellow dotted lines) with distances measured in Å (red).

Figure 3. Mass spectrum recorded for SemiSWEET and the effect of cardiolipin on the monomer dimer equilibrium.

Mass spectrum of SemiSWEET following incubation with cardiolipin (CDL, purple head-groups). Plot of CDL concentration (mg ml^-1) versus the percentage of monomer or dimer observed in mass spectra at various CDL concentrations (bars represent n=5 data points denoted with black dots, the error bars denotes the standard deviation).

Figure 4. Comparison of mass spectra of NhaA with NapA and plot of stabilities of transporters studied here with G-protein coupled receptors.

a, Mass spectrum of NhaA (green/grey spheres), liberated from C₈E₄ micelles, reveals binding of cardiolipin (CDL, purple head-groups) and phospholipids (PL, blue head-groups) to the intact NhaA dimers. MD simulation of NhaA (green/black rods) in an E. coli lipid bilayer (inset) reveals interfacial CDL binding (orange/purple spheres). Mass spectrum of dimeric NapA (right), liberated from C₈E₄ micelles, shows NapA dimers without lipid binding (blue/brown spheres). b, Oligomeric stability scale (purple) annotated with crystal structures of proteins studied here (above) and GPCRs (below). Buried surface area is shown in parenthesis and number of salt bridges (SB) is shown for NapA. PDB IDs: 2A65 (LeuT), 4QND (SemiSWEET), 4AU5 (NhaA), 4BWZ (NapA), 4GPO (β1 adrenergic receptor), 4DKL (μ-opioid receptor), 4DJH (κ-opioid receptor).