*3.1. CitS*

The structure of the CitS transporter from *Salmonella enterica* reveals the presence of a lipid, assigned as phosphatidylethanolamine (PE) on the edge of the protein [64]. It is not clear if this lipid has any specific role in CitS structure and function, but the lipid molecule is only detectable on one of the two protomers in the CitS dimer. Given that the two protomers are in different conformational states it is possible that binding of the lipid is conformationally specific.

#### *3.2. UapA and ScBOR1p*

In some cases, lipids might be associated with a membrane protein but too disordered to be visible in the crystal structure. This was the case with UapA, a xanthine/uric acid transporter from *Aspergillus nidulans*, whose structure was determined as a closely associated, functionally relevant dimer with no visible lipids [65]. Native mass spectrometry (MS) analysis is proving to be a very powerful method for exploring membrane protein– lipid interactions [51,66], and this approach revealed that the UapA dimer isolates in the detergent dodecylmaltoside in complex with membrane lipids [26]. Further lipidomics analysis identified that UapA co-purifies with the membrane lipids, phosphatidylinositol (PI), phosphatidylcholine (PC) and PE. Loss of these PLs results in dissociation of the UapA dimer into the monomeric state, with the dimer recoverable through the addition of exogenous PE and/or PI. Molecular dynamics simulations predicted the location of a lipid binding site made up of three Arg residues (R287, R478, R479) at the dimer interface and on the intracellular membrane leaflet. Subsequent mutagenic and MS analyses indicated that substitution of these three Arg residues caused loss of function and resulted in protein

that was almost exclusively in the monomeric form. Addition of exogenous lipid to the mutant lacking the binding site was unable to recover the dimer form, strongly indicating that binding of lipids to this site in the wild-type protein is key in the formation and maintenance of the physiological dimeric state [26]. This research highlighted that the PLs were essential for functional dimer formation and since the crystal structure of UapA is a dimer, PLs must be present in the crystals of UapA, albeit too disordered to be detectable in the final structure [65].

UapA is structurally and mechanistically related to other transporters from the solute carrier (SLC) 4 and SLC26 families, including the BOR proteins, boron transporters. The BOR protein from *Saccharomyces cerevisiae*, ScBOR1p, isolates as a monomer in both dodecylβ-D-maltoside (DDM) and Triton X-100 [27], but lipidomics analysis reveals that it copurifies in the presence of PI, PE, PC and phosphatidylserine (PS). As with UapA, addition of exogenous lipid to delipidated ScBOR1p causes the monomers to associate into dimers as revealed by native MS. A similar lipid binding site was predicted at the intracellular side of the dimer interface of ScBOR1p, from a model of the protein based on the crystal structure of *Arabidopsis thaliana* BOR1 [67]. Mutagenesis of the lipid binding site in ScBOR1p prevents lipid dependent dimer formation but does not abolish transport function, indicating that the dimer is not critical for function and highlighting that although there are clear similarities in the interactions between UapA and ScBOR1p and membrane lipids, the precise functional outcome of that interaction differs between the two proteins [27].

#### *3.3. Monoamine Transporters, hSERT and hDAT, and the Homologue LeuT*

Differences in relatively related transporters and their interactions with lipids are also seen in the serotonin (hSERT) and dopamine (hDAT) transporters, both monoamine transporters. There is strong support for both transporters being oligomeric in the native membrane, with the hSERT oligomers stabilised by phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) binding [40]. However, whilst there is clear evidence of binding of PtdIns(4,5)P2 to the N-terminus of hDAT and a suggested role in amphetamine-induced dopamine efflux through the hDAT [41], stability of the hDAT dimers is independent of PtdIns(4,5)P2 as assessed by single molecule fluorescence microscopy before and after enzymatic depletion of PtdIns(4,5)P2 in the membrane [68]. Crystal structures of both the drosophila DAT [69,70] and the hSERT transporter [71] identify putative cholesterol-binding sites. Cholesterol is important for the functioning of hSERT. Recent analysis indicates that mutation of a cholesterol-binding site in hSERT or depletion of membrane cholesterol results in the transporter preferentially adopting the inward facing conformation which in turn reduces transporter activity [8,9]. Conversely, mutations in the cholesterol binding site that favour cholesterol binding cause the transporter to preferentially adopt an outward facing conformation [9]. Thus, association and dissociation of cholesterol at a key site in the protein may play an essential role in regulating hSERT activity. Given the fact that similar effects of cholesterol binding on transporter conformation are reported for hDAT [72,73], it is possible that cholesterol plays a similar regulatory role in hDAT.

A recent publication from the groups of Michael Landreh and Carol Robinson explored the concept of annular lipids that provide the hydrophobic environment essential for maintaining the overall structure of the protein [74] versus specific lipids in more detail [75]. They developed a method which exploits the fact that loosely bound, annular lipids on the surface of a protein are more prone to exchange for detergent molecules than closely associated lipid molecules. Following detergent exchange, the protein is analysed by native MS which detects changes in the abundance of bound lipid. The results of this study revealed that the bacterial presenilin homologue forms only weak interactions with annular lipids whereas LeuT, a bacterial homologue of the monoamine transporters, binds both specific lipids at a dimer interface and annular lipids on the periphery [75]. Additional analysis has indicated a role for CL in the oligomerisation of LeuT [51], a lipid also implicated in allosteric regulation of the bacterial lipid II exporter, MurJ [52].

#### *3.4. The Betaine Transporter, BetP*

Although it can be difficult to obtain structural insights into transporter-lipid interactions as a result of both the loss of lipids and the poor structural resolution of the lipid molecules, it is possible. One example is the bacterial transporter, BetP, involved in osmoregulation, which crystallised in complex with lipids whose density was clearly discernible in the high resolution (2.7 Å) structure. The lipid density was assigned as eight palmitoyl-oleoyl phosphatidyl glycerol (PG) molecules [36], with seven of the PG molecules located on the intracellular side of the protein and one located on the extracellular side. Five of these lipids mediate protomer–protomer interactions within the BetP trimer (Figure 1A) playing a fundamental role in oligomer formation. The remaining three lipid molecules, including the lipid on the extracellular side of the protein, are more loosely associated with the periphery of the protein (Figure 1B,C) and are likely to be annular lipids. The lipids bound to BetP are associated with regions known to be involved in conformational changes associated with transport activity and transport regulation, strongly indicating that in addition to the quaternary structure of the protein, lipid binding is also critical for function [36].

**Figure 1.** Crystal structure of the trimer of BetP in complex with the lipid (PDB: 4C7R [36]). The individual protomers are shown as green, bright pink and blue ribbons. The lipids key in trimer formation are shown in sphere representation with yellow carbon atoms. The annular lipids bound to the periphery of the protein are shown in sphere representation with cyan carbon atoms. ( **A**) The protein–lipid complex is shown from the intracellular side of the membrane. (**B**,**C**) The protein–lipid complex in two different views looking through the membrane to illustrate the different locations of the peripherally bound lipids.

#### *3.5. The Cationic Amino Acid Transporter, GkApcT*

The presence of lipid, in this case cholesterol, was essential for crystallisation of a cationic amino acid transporter homologue from the thermophilic bacterium, *Geobacillus kaustophilus,* GkApcT [76]. A cholesterol molecule sits in a pocket formed by interactions between the GkApcT and another single transmembrane domain protein, MgtS, stabilising this interaction. Clearly this is a very interesting lipid dependency since bacteria do not produce cholesterol. The authors of the study sugges<sup>t</sup> that the cholesterol may be a functional replacement for a group of chemically similar lipids, called the hopanoids, found in some bacteria [77]. Whilst the precise role of the potential hopanoid interaction in vivo is not clear, it is possible that the lipid plays a role in transporter regulation as described above for cholesterol and the monoamine transporters.

#### *3.6. The Major Facilitator Superfamily Sugar Transporters, LacY and XylE*

An exciting new development in understanding the structural and functional implications of membrane protein–lipid interactions is hydrogen–deuterium exchange mass spectrometry (HDX-MS). HDX-MS defines the solvent accessibility of different regions of a protein by monitoring the exchange of hydrogen to deuterium; the exchange reaction depends on intrinsic protein motions [78]. H-bonding networks greatly reduce the rate and efficiency of HDX, whereas highly dynamic regions typically undergo a higher level of HDX [79]. In a recent study, Martens et al., [28] explored the conformational state of two bacterial sugar transporters, LacY and XylE, that had been purified in detergent and

then reconstituted into nanodiscs incorporating a mix of PC, PG and CL or PE, PG and CL. Nanodiscs are membrane mimetic systems formed from a membrane scaffold protein, exogenous lipids and the target protein, with the scaffold protein wrapping around the complex of lipids and protein. This arrangemen<sup>t</sup> shields the protein from aqueous solution and produces a more native-like environment than detergent micelles whilst also allowing ready variation of the lipid composition [80,81]. The results of the study on LacY and XylE revealed that in the presence of PE both transporters were preferentially in the inward-facing conformational state. The ability to control the lipid composition surrounding isolated protein through nanodisc reconstitution is a powerful means of exploring the role of individual lipids; however, it does suffer from the disadvantage that the protein is isolated initially in detergent and then lipids added. An alternative approach is to use the SMALPs together with HDX-MS, allowing analysis of the conformational dynamics of membrane proteins encapsulated in their native membranes as described for the bacterial rhomboid protease, GlpG [82]. Here, the authors were able to alter the lipid composition at the level of the bacterial membrane by varying the expression strain or the expression temperature. They found that the conformational flexibility of the protein differed depending on the lipid composition. Such approaches have major potential for exploring the protein–lipid relationships of other classes of membrane proteins. Additionally, researchers are developing direct methods, performing MS on proteins removed directly from the membrane without the need for any extraction agents [83]. This technology is still in the early stages, but it has potential to provide information on the direct physiological interactions of membrane proteins with membrane lipids as well as protein–protein interactions.

#### **4. ATP Dependent Pumps and Transporters**

Lipids are also crucial for ATP dependent pumps and transporters. The Na+, K+ ATPase, for example, was crystallised in complex with a molecule of cholesterol bound between the α and β subunits [84]. The precise role of the cholesterol bound to this protein has been subject to debate, possibly due to the non-physiological conditions used for study of the effect of the lipid. However, recent research uses the addition of methyl-βcyclodextrin to deplete cholesterol from membrane fragments while keeping all the other membrane components intact [10]. This study revealed that Na+, K+ ATPase is less active when there is a lower amount of cholesterol in the membrane and suggests that this is due to less efficient transition between different conformational states critical for the transport cycle.

Cholesterol is also important for the ATP binding cassette (ABC) transporter, PgP. PgP is an extremely important example from this family responsible for multi-drug resistance in many types of cancers [85]. The biochemical relationship between PgP and cholesterol appears to be multi-faceted, involving possible roles for cholesterol in ATPase activity, in modulating binding affinity of some transported substrates, as well as a possible direct function for PgP in cholesterol trafficking [11]. Although there is no direct structural information available for cholesterol binding to the PgP, recent MD simulations have identified several putative cholesterol binding sites and have suggested that the cholesterol cluster specifically on one side of the protein interacts with TM domain 1 [86]. The study also revealed that cholesterol flipping from one leaflet of the membrane can occur along the surface of the protein [86].
