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...

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as, hypertension, heart failure and epilepsy and they are well-established drug targets2. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli1,3, where both EM and crystal structures are available4-6. NhaA is made up of two distinct domains, a Core domain and a Dimerisation domain. In the NhaA crystal structure a cavity is located between the two domains providing access to the ion-binding site from the inward-facing surface of the protein1,4. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, where a conformational change is thought to occur7. To date, the only reported NhaA crystal structure is of the low pH inactivated form4. Here, we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the Core and Dimerisation domains are in different positions to those seen in NhaA and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to directly coordinate ion-binding1,8,9, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the Core domain, some 20° against the Dimerisation interface. We conclude that despite their fast transport rates of up to 1500 ions/sec3, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

A dimeric structure of the sodium–proton antiporter NhaA provides insight into the roles of Asp163 and Lys300 in the transport mechanism.

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Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary-active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid-binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild-type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter-domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator-type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large-scale conformational changes within the membrane.

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Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na + + + /H + + + antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na + + + /H + + + antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by ∆pH, but no longer respond to either an electrochemical potential (Ψ) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na + + + /H + + + exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na + + + /H + + + antiporters.secondary active transporters | proton transport | membrane protein | Na + /H + exchangers | energetics