The role of interfacial lipids in stabilizing membrane protein oligomers

Abstract

Oligomerisation of membrane proteins in response to lipid binding plays a critical role in many cell-signaling pathways 1 but is often difficult to define 2 or predict 3. Here we develop a mass spectrometry platform to determine simultaneously presence of interfacial lipids and oligomeric stability and discover how lipids act as key regulators of membrane protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins revealed 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 (CDL) deficient Escherichia coli, abrogated dimer formation. Molecular dynamics simulation revealed that CDL acts as a bidentate ligand bridging across subunits. Subsequently, we show that for the sugar transporter SemiSWEET from Vibrio splendidus 5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesised that lipids would be essential for dimerisation of the Na + /H + antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for substantially more stable, homologous NapA from Thermus thermophilus. We found that lipid binding is obligatory for dimerisation 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 Competing Financial Interests:The authors declare no competing financial interest Data Availability. The raw data for Figure 1 is provided in the Supplementary Table 1. All other data are available upon request. Europe PMC Funders GroupAuthor Manuscript Nature. Author manuscript; available in PMC 2017 July 19. Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including GPCRs.The recent surge in structure determination of membrane proteins is providing details of protein-lipid binding 6 and yielding insight into the regulatory roles of lipids 7,8. The advent of mass spectrometry (MS) methods for characterising membrane proteins, individually 9, within interactomes 10, and in intact assemblies 11, is adding new information to potential roles of lipids inducing conformational changes 12, contributing to activity and modulating drug efflux (reviewed in 13). The role of lipids towards maintaining the oligomeric state of membrane proteins has however remained widely debated. To understand this phenomenon we performed a bioinformatics analysis of all the α-helical oligomeric transmembrane proteins with known structures. To gauge their relative stability, we ranked these olig...

Solvent Mediation of Peptide Conformations: Polyproline Structures in Water, Methanol, Ethanol, and 1-Propanol as Determined by Ion Mobility Spectrometry-Mass Spectrometry

“…However, non-native folds are typically transient in nature [3, 5, 7] and sensitive to the local environment. For example, interactions with solvent, [8–10] small molecules [11–13] (e.g., osmolytes, drugs, lipids) and extreme cases where the polypeptide is imbedded or interacting with membranes [14, 15] can modulate biomolecular structure, increasing the complexity of the folding process. Mass spectrometry, especially “native MS”, has emerged as an ideal biophysical tool to study biomolecular structure and interaction [16–19].…”

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

“…We investigated further the possibility of detergent screening on the DESI stage using the sugar transporter semiSWEET from Vibrio splendidus 21 since it is extremely sensitive to its detergent environment. Previous MS experiments established that this transporter exists in an entirely monomeric form in DDM and exhibits a monomer–dimer equilibrium in the detergent (C8E4) 22. Depositing semiSWEET on the DESI stage in DDM (Figure 2 d) we then added C8E4 (0.5 % w/v) directly to the desorption buffer.…”

Mass spectrometry-enabled structural biology of membrane proteins

“…It is becoming more and more apparent (from EM, X-ray crystallography and MS studies [22,96,[98][99][100]) that there is an intimate relationship between MPs and the surrounding lipid environment, whereby the binding of key lipids is important for maintaining MP structure and function [98]. Detergents are a poor mimic of the native bilayer, may not permit the retention of MP-lipid interactions [101], and in many cases disturb the correct, functional oligomeric state of MP assemblies [90,95,96].…”

“…Intriguingly, MS has been used to show that delipidation of certain MP complexes results in their destabilization and dissociation into their constituent components [22]. This is related to the strength of the oligomerization interface, with complexes comprising interfaces with low buried surface areas and few salt bridges requiring lipid binding for stabilization [22]. Additionally, the binding of lipids that have been carried through purification protocols has been observed in native mass spectra for several MPs [20,21,89,129].…”

“…Native MS methods can also be used to detect lipid, drug, or other molecules binding to MPs. Intriguingly, MS has been used to show that delipidation of certain MP complexes results in their destabilization and dissociation into their constituent components [22]. This is related to the strength of the oligomerization interface, with complexes comprising interfaces with low buried surface areas and few salt bridges requiring lipid binding for stabilization [22].…”