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mPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations [1]

['Alexandra O. M. Holmes', 'School Of Biomedical Sciences', 'Astbury Centre For Structural Molecular Biology', 'University Of Leeds', 'Leeds', 'United Kingdom', 'Adrian Goldman', 'Research Program In Molecular', 'Integrative Biosciences', 'University Of Helsinki']

Date: 2022-11 

Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.

In this work, we demonstrated conservation of lipid-interaction sites on proteins from two species that deviated from their evolutionary common ancestors a long time ago, using the membrane-integral pyrophosphatases from a thermophilic bacteria species and a plant species. We further showed that these sites are preserved in homology modelled proteins of the same family. This retention of a common lipid interaction profile or “fingerprint” and our ability to predict this for other proteins in this family may indicate that they are more integral to protein function than previously thought. By identifying lipid interactions that may act to stabilise the protein structure, these properties could be exploited to solve protein structures, and the interfacial site’s potential involvement in inter-subunit communication may be useful for further investigation of the catalytic cycle of this clinically relevant membrane protein family.

Funding: This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) DTP fellowship (BB/M011151/1) (to AH). AG was funded by a BBSRC grant BB/T006048/1 and a grant from the Academy of Finland, grant number 1322609. This project also made use of time on ARCHER supercomputer granted by the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim ( www.hecbiosim.ac.uk ), which is supported by the Engineering and Physical Sciences Research Council (EPSRC; grant no. EP/R029407/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we examined the interactions and dynamics of three mPPases structures in model lipid bilayers via multi-scale MD simulations. Our results suggest that mPPases form an anionic annulus in the membrane and possess specific anionic lipid binding sites at the dimer interface and the distal regions of the protein. These protein-lipid interactions are conserved between mPPases from different species with differing pumping specificities, which may suggest that these are a general property of mPPases.

In recent years, molecular dynamics (MD) simulations have played a role in uncovering and studying membrane protein-lipid interactions [ 18 ]. These interactions can have multiple effects on the protein of interest, for example modulating stability [ 19 ], assisting conformational changes [ 20 ], oligomerisation [ 21 , 22 ] and large-scale protein organisation [ 23 ]. MD simulations represents a robust way to identify putative lipid binding sites which can be refined through further simulations or in tandem with other methods [ 18 ].

These 3D structures have facilitated understanding of the mechanism by which mPPases perform hydrolysis and ion pumping. However, there is conflict surrounding the order of these two events [ 15 , 16 ] and more recently, data have been published indicating that the mechanism is more complicated than previously thought [ 10 , 17 ]. There is also a dispute around how the mPPase subunits operate with one another, which may be explained by the environment of the protein: lipidated [ 17 ] or detergent-solubilised [ 10 ], and so could indicate protein-lipid interactions [ 1 ]. Despite evidence that the lipid environment may have a role in regulating the function of mPPases, the interaction of mPPases with their lipid environment is still unknown.

A) the Tm-PPase resting state crystal structure (PDB: 4AV3) viewed from above to demonstrate the concentric circles of TMH of each subunit (outer ring in cyan and inner in pink) and the TMH contributing to the interface with the adjoining subunit (grey surface representation). B) The atomistic simulation box where the protein subunits are each coloured in cyan and pink and shown as Cartoon representation, the POPE (80%) and POPA (20%) bilayer is shown as lines representation and the water particles are grey points, and the ions (NaCl 150 mM) are shown as yellow VDW particles.

Crystal structures revealed that mPPases exist as homodimers, where each subunit is composed of 16 transmembrane helices (TMH) [ 7 – 11 ]. A single subunit is formed by two concentric rings of TMH: the inner ring (TMH 5, 6, 11, 12, 15 and 16) makes up the 4 catalytic regions: the catalytic centre, the coupling funnel, the ionic gate and the exit channel, while the outer ring (TMH 1, 2, 3, 4, 7, 8, 9, 10, 13, 14) forms the subunit-subunit interface and the membrane-facing surface of the protein ( Fig 1A ). Despite their common structure, seven different mPPase subfamilies have been functionally characterised [ 12 – 14 ]. In short, mPPase catalytic activity is either K + dependent or K + independent and the pumping specificity is either H + only, Na + only, dual H + /Na + or H + regulated by Na + . Of these subfamilies, only the 3D structures of the K + -dependent H + -PPase from Vigna radiata (Vr-PPase) [ 7 ], and the K + -dependent Na + -PPase from Thermotoga maritima (Tm-PPase) [ 8 – 10 ] have been resolved to high resolution.

Membrane integral pyrophosphatases (mPPases) are a family of membrane proteins responsible for coupling the hydrolysis of the pyrophosphate (PP i ) phosphoanhydride bond to the pumping of a cation across the membrane [ 1 ]. This allows mPPases to both remove excess PP i from the cytoplasm, and to generate a membrane potential. mPPases are found in all kingdoms of life, excluding fungi and multicellular animals [ 2 ]. Due to this, they are validated selectively toxic drug targets against a variety of protozoan and bacterial pathogens [ 3 – 6 ] and reducing the function of mPPases via novel inhibitors may play a role in combatting these infectious pathogens.

The association of the anionic lipids at the WT protein interface was maintained throughout the 250 ns simulations, as was predicted with the CG residence times, and interactions with all the proposed lysine and arginine residues at the mPPase protein interface and distal interaction sites were retained. Analysis of the simulations revealed interacting residues that had not been predicted by the initial CG simulations, such as with Y 9.64 (383), Y 13.45 (561) and Y 13.51 (567) for Tm-PPase ( S13 Fig ). However, these appear to only interact a proportionately small amount of the time compared to the lysine and arginine residues. In all these simulations there appeared to be no differences between the two interfacial binding sites in each mPPase or in the dynamics between the mutated and WT proteins throughout the atomistic simulations ( S8 and S9 Figs ). However, the RMSD of the proteins in the systems TmPA20_DSM, TmPG20_DSM and CpPS20 did not appear to flatten throughout the 250 ns simulations, which may be due to the high flexibility of the 5–6 and 13–14 loops, as seen in the RMSF analysis ( S8 Fig ) .

The effect of lipid binding on the dimeric arrangement or stability of the protein could not be assessed through these coarse-grained (CG) simulations, as the elastic network between the protein monomers maintained the protein-protein interface and conformational state, while allowing flexible regions to move ( S8 Fig ). Therefore, to refine the protein-lipid interactions at these sites and identify any effect the lipid binding had on protein structure and dynamics, the final coordinates of the WT or DSM Tm-PPase, Vr-PPase and Cp-PPase systems were converted to an AT representation (see Methods ). Frames were chosen where at least one anionic lipid was bound to all of the anionic lipid interaction sites so that their binding could be better understood at an atomistic resolution.

The electrostatic profile as calculated by APBS and structural arrangement of the A-B) Tm-PPase, C-D) Vr-PPase and E-F) Cp-PPase protein interfacial anionic lipid binding site with POPA (green sticks) or PIP 2 in the case of Vr-PPase bound and the interacting lysines or arginines shown as sticks. The snapshots are the final frame of one of the CG simulations (at 5 μs of simulation) converted to an atomistic representation. G) The distal interaction site of Tm-PPase, and H) the arrangement of a DM detergent molecule (green sticks) in the crystal structure of Vr-PPase (PDB: 4A01).

To examine whether there was any synergy in the binding of lipids in the lipid binding sites identified above, we performed in-silico mutagenesis of the four positively charged residues in the interfacial site to alanine. Analysis of the lipid density following identical simulation to the wild-type (WT) proteins showed that when a single or double site mutation (DSM) was made, the anionic lipid binding was lost, but binding at the intact site remained. This, therefore, indicates that our simulations do not demonstrate any cooperativity between the binding sites. This confirmed that the anionic lipid binding at these sites was due to specific interactions with the protein, rather than a stochastic effect, and that the binding is independent at each site. To assess whether this was due to the properties of the protein surface in these areas, electrostatic analysis of the proteins was performed and revealed that positively charged regions matched the location of the anionic binding at the interfacial and distal regions of the protein ( Fig 4 ). The electrostatic profiles were very similar between the three mPPases in this study, as well as the arrangement of their binding site residues ( Fig 4 ).

Interestingly, the distal and interfacial sites of Cp-PPase better resembled that of Tm-PPase, despite being modelled with reference to the Vr-PPase structure, both in structural arrangement and lipid occupancy and average residence time. The interfacial site accommodates up to 2 anionic lipids at a time and the distal site could accommodate up to 4 ( S6 Fig ), despite being comprised of only 7 positive residues, similar to the interactions seen with Tm-PPase in the same bilayer systems. However, the sites have shorter average residence times than in the other mPPase simulations, with only 0.1 μs for the distal site, primarily contributed to by K 2.46 (51), R 2.47 (52), K 3.59 (101), R 7.62 (290), and the interfacial site had an average residence time of 0.2 μs.

As in the other systems, Cp-PPase formed an anionic lipid fingerprint in the membrane ( S3 Fig ), where specific interactions formed between the lipids and positively charged residues at the distal site (R 1.56 (23), K 1.60 (27), K 2.46 (51), R 2.47 (52), K 3.59 (101), R 7.62 (290), K 7.64 (292)) and at the interfacial site (K 9.69 (361), K 9.73 (365), R 13.52 (543), R 14.48 (584)). The interfacial site was made up of two residues from each protein subunit and Cp-PPase displayed a preference for POPA over the anionic lipids POPG or POPS in the mixed simulations. This supports our previous hypothesis that binding of anionic lipids to these two types of interaction site may be a general property for all mPPases.

Clostridium species are predicted to contain POPG, POPE, POPS and cardiolipin, in addition to diradylglycerols. For this reason, we simulated this structure in the same bilayers as Tm-PPase. This also enables better comparison of our results between the two systems. The Cp-PPase simulations were run with 20% POPA, POPG or POPS or an equal anionic lipid mix (10% each). As well as being a crystallographic target, Cp-PPase is also a different subtype of mPPase compared to Tm-PPase and Vr-PPase; this allowed us to examine whether the anionic interactions identified for two different mPPases are retained in other subfamilies.

Our results above suggest the two structurally characterised mPPases induce an anionic lipid fingerprint in model membranes. As other mPPase studies have also indicated that lipid binding could be of interest for functional and stabilising studies [ 17 , 30 ], we modelled the Na + /H + -PPase from C. leptum (Cp-PPase; see Methods for details) to examine whether the anionic lipid fingerprint is found in other members of this family. Despite producing two high quality models from both Robetta and AlphaFold2 with significant similarity (1.05 Å RMSD), the Robetta homology model (reference PDB ID: 5GPJ) was chosen for this study as the arrangement of the distal helices was more in keeping with those seen in Tm-PPase and Vr-PPase structures ( S10 Fig ).

Despite having similar structural arrangements of the interfacial and distal sites as in Tm-PPase, the interactions between the lipids and the protein were somewhat different in Vr-PPase. The interfacial site appears to be smaller and only accommodates one anionic lipid at a time for the majority of the 20 μs of simulation time, with a second lipid interacting with the binding site very rarely. However, when there are two lipids interacting with the binding side, one lipid interacts with residues K 13.48 (595) and K 14.48 (632) that are at the centre of this binding site and the second lipid interacts with one of the outer residues of the site (K 10.49 (457) and K 14.40 (640)) ( S2Q–S2R Fig ). Despite this, the average residence time in the interfacial site was longer than that seen for Tm-PPase, at 0.51 μs ( S5 Fig ). The residence time analysis at each protein residue revealed that there was a lot of variability in the residence times of the different residues in the binding site, and residue K 4.37 (181) (1.25 μs residence time) had the most prolonged interactions with lipids in this site compared to the others ( S5 Fig ). The distal interaction site also appeared to be smaller for Vr-PPase compared to Tm-PPase, as it formed contacts with a maximum of 3 lipids, but primarily 1 or 2. The reduced occupancy may be due to the increased size of the PIP 2 head group and availability of negative charge to form interactions with, thereby reducing the availability of binding site residues or the tilting of TMH 13 and 14 towards the interface compared compressing the site compared to Tm-PPase in the resting state [ 9 ].

As these simulations contained additional anionic lipids compared to the systems with Tm-PPase, we were able to study the preference of Vr-PPase for anionic lipids in a more realistic membrane. All of the anionic lipids formed interactions with the interfacial and distal interaction sites, but our analysis suggests that interactions between Vr-PPase and PIP 2 were highly favoured over the neutral and other anionic lipids ( Fig 3B ). Of the palmitoyl-oleoyl phospholipids, POPA was the favoured lipid over both POPG and POPS, despite its low representation in the membrane (1%) ( S2A–S2H Fig ). Therefore, the interactions with PIP 2 and POPA were used to understand the properties of the Vr-PPase sites ( Fig 3C ).

The interactions between Vr-PPase and a realistic tonoplast membrane represented through A) the average density of the phosphate particles of the anionic lipids (PIP 2 , POPG, POPS and POPA) calculated using a combined trajectory fitted around the protein, B) the normalised number of contacts between the lipids in the bilayer and the residues of the protein, alongside C) a representation of the contacts on the 3D protein structure of PIP 2 (orange) and POPA (green).

As we have more information about the V. radiata tonoplast membrane, simulations of Vr-PPase were performed in bilayers resembling this membrane (29% cholesterol, 25% POPC, 17% POPE, 17% ceramide hexoside, 6% PIP 2 , 3% POPG, 2% POPS and 1% POPA) [ 29 ]. The four anionic interaction sites seen with Tm-PPase were present at the interfacial and distal regions of the Vr-PPase protein ( Fig 3A ), where lysine and arginine residues formed the distal site (K 1.60 (35), K 1.62 (37), K 1.67 (42), K 2.50 (94), K 3.59 (162), R 4.33 (177), K 4.37 (181), R 4.44 (188), K 8.41 (352), K 8.52 (363)). However, unlike Tm-PPase, the distribution of residues forming the interfacial site was uneven, as one from one subunit and three from the other contributed to forming interactions with the anionic lipids (K 10.49 (457), K 13.48 (595), K 14.40 (632), K 14.48 (640) for Vr-PPase). These simulations were run with 323 K temperature as in our previous simulations, but protein-lipid interactions were very similar to the same simulation in which the temperature was 310 K ( S7 Fig ).

To examine whether the anionic lipid fingerprint identified above was Tm-PPase specific or if it also occurs in mPPases in other species and in other membranes, we also performed simulations using the crystal structure of Vr-PPase. Like Tm-PPase, Vr-PPase is structurally well-characterised but mesophilic rather than thermophilic and, as there are no resting state Vr-PPase structures available, the relaxed product bound state (PDB: 5GPJ) was chosen for simulation to be the most comparable to the Tm-PPase findings.

Contact analysis and PyLipID [ 28 ] were used to understand the lipid occupancy, exchange and residence of the distal and interfacial sites. The distal site was able to accommodate more lipids, with 2 lipids bound the majority of the time, rarely increasing to 3 or in some cases 4 for short periods ( S1V–S1W Fig ). Residues R 2.38 (43), R 3.63 (100) and K 8.40 (311) had the highest lipid residence times (1.41 μs, 0.23 μs and 0.13 μs, respectively) ( S4 Fig ) and were found to primarily coordinate 2 or 3 lipids in the site, with the 4 th lipid briefly interacting with other residues in the site. The interfacial site was able to accommodate fewer lipids at any time, usually being occupied by one or occasionally two lipids. Rarely a third lipid would interact with the site, but this was with only one of the outer residues (typically K 14.48 (609)), rather than being coordinated within the site. This is consistent with the size of the sites, as the interfacial site is much smaller than the distal site, and is situated in an interfacial cleft, causing the inner residues to be less accessible for contacts. Additionally, the rate of lipid exchange was higher in the interfacial site than the distal site, with residence times averaging 0.28 μs for the four interfacial residues ( S4 Fig ).

Analysis of the lipid contacts revealed that POPA interacted more frequently with Tm-PPase compared to POPG and POPS at 20% anionic lipid bilayer content ( Fig 2D–2I ). To examine further the more frequent binding of POPA lipids, we also performed a simulation in which the bilayer contained an equal anionic lipid mix (10% each of POPA, POPG and POPS). In this simulation, POPA interacted up to 43-fold or 84-fold more frequently than POPG or POPS, respectively, supporting our previous observation that POPA interacts more with Tm-PPase compared to other anionic lipids ( S1D Fig ) . Therefore, further characterisation of the Tm-PPase lipid binding sites in this work was performed using the system that contained Tm-PPase with 20% POPA lipids in the bilayer (TmPA20 in Table 2 ) system, but it was found that, for all of the parameters assessed, the interactions with POPG and POPS were reduced compared to those with POPA.

Analysis of the interactions between the Tm-PPase and the anionic lipids allowed us to identify lipid binding sites through contact analysis and lipid density. These binding sites were identified using the top 5% of interacting residues from the contact analysis. This analysis revealed four symmetrical anionic lipid binding sites at the dimer interface and the “distal regions” of the protein ( Fig 2B and 2C ). Nine positive lysine and arginine residues (R 1.60 (27), K 1.61 (28), R 2.38 (43), K 3.59 (96), R 3.63 (100), R 3.67 (104), K 4.40 (120), K 8.40 (311) and K 8.41 (312)) [note that the Ballesteros and Weinstein numbering system is used [ 13 , 27 ] alongside traditional residue numbering] formed each of the distal interaction sites. The residues involved in the dimer interface sites were four positive lysine residues (K 9.70 (389), K 10.49 (415), K 13.52 (568) and K 14.48 (609)), two of which were located in one subunit and two in the other subunit.

The contacts between the lipid bilayer and Tm-PPase for model bilayers consisting of 80% POPE and 20% anionic lipid represented by A) normalised lipid contacts with both POPE and POPA/G/S and the residue number of the protein, B) the density of the PO4 bead of the CG anionic lipid around the protein, where the trajectory has been fitted to the protein backbone and C) the contacts of the PO4 atom represented on the 3D structure of the protein with a colour scale from white to green, for POPA, D-F) blue for POPG, and G-I) red for POPS. The lipid contacts were normalised by dividing the number of contacts of each residue with the length of simulation and the number of lipid species.

We first performed simulations with the 3D structure of the Tm-PPase, due to the extensive structural characterisation of the protein over the last decade [ 8 – 10 ]. The Tm-PPase structure was inserted into bilayers containing POPE and POPA, POPG or POPS molecules ( Table 1 ) ( Fig 1B ). Little is known about the specific compositions of the T. maritima native lipid bilayers [ 24 – 26 ] and so palmitoyl-oleoyl phospholipids were considered an appropriate proxy. Following 5 μs of simulation, the normalised contacts of the protein with the anionic component in the inner leaflet of the bilayer were higher for all residues compared with the other lipid components, suggesting that the anionic lipids POPA, POPG and POPS interacted preferentially with Tm-PPase in the cytoplasmic leaflet compared to the zwitterionic POPE lipids ( Fig 2 ) . Titration of the concentration of each anionic lipid, (40%, 20% and 30% (made up of 10% of each anionic lipid) of the overall lipid concentration) demonstrated that this annulus was retained in all concentrations with ~20 lipids interacting with the protein in each concentration ( S1 Fig ). Therefore, the 20% anionic lipid bilayers were chosen for further experiments, so selective interactions could be distinguished.

Discussion

In this study, we have created atomistic models of three different mPPases in modelled membranes. Our work with Vr-PPase represents a realistic model of an mPPase in a complex bilayer that mimics its native environment. Our simulations have shown that mPPases inserted into a bilayer create an anionic lipid fingerprint around them with preferential interactions with anionic lipids at two distinct sites. In mPPases, two pertinent studies of catalytic asymmetry and inter-subunit communication reported different changes in PP i affinity at the second monomer following binding of substrate at the first. One of the major differences in these experiments was whether the mPPases were in a lipid [17] or detergent [10] environment. These data suggest that lipid binding could have a modulatory role in inter-subunit communication and catalytic asymmetry, and delipidation through solubilisation and purification procedures thereby influences mPPase function.

In this investigation, mPPases were found to interact preferentially with anionic lipids compared to neutral lipids. This interaction appears to be a result of the charged surface of the protein, as demonstrated by the electrostatic profile analysis and loss of interaction following mutation and loss of charge. In addition to this, the pronounced preference for POPA in Tm-PPase may be due to its increased negative charge compared to the POPG and POPS lipids (-2 vs -1) or due to its similarities with the T. maritima native dietherglycerol phospholipids lipids, as both are anionic and do not possess headgroups [26]. This may also be the case for the Cp-PPase preferential interactions with POPA. Non-specific lipid interactions have been proposed to affect protein function. For example, cardiolipin binding to UraA (H+-uracil symporter) [31], cytochrome bc 1 (respiratory chain complex) [32] and SecY (proton translocon) [33] is thought to attract positive ions for pumping. It is unclear whether this functionality exists for other anionic lipids, as it is believed that this proton trap capability comes from the presence of two phosphate groups with different pK a values [34]. However, more recent studies suggest the pK a values are similar for each cardiolipin phosphate [35,36], so potentially this functionality is applicable to the single-phosphate anionic lipids in this investigation. Therefore, the anionic lipid clustering around the mPPases could serve to attract the pumped cations and the Mg 2 PP i for catalysis.

The observed anionic interactions were primarily localised to the cytoplasmic leaflet (Figs 2 and S12). Charged regions at the cytoplasmic side of proteins were shown to have a role in membrane protein insertion into the bilayer [37]. However, the clustering of anionic lipids to these areas and the specific interactions identified in this study may also play role in the function and stability of mPPases. Similar interactions between the lysine and arginine residues of the identified binding sites and the anionic lipid headgroups were also found in other proteins [38–40], in which the lipid interactions had marked effect on protein stability, function or dynamics. Additionally, it was more recently demonstrated for several oligomeric membrane-integral transporters that interfacial lipids play a crucial role in oligomerisation and stability [21,22]. Lipids are also considered capable of stabilising transient conformational states [20]. The need to stabilise alternative states of the catalytic cycle and different members of the mPPase family for structural studies means that MD-identified lipid interactions may be a key part of achieving structural information, particularly as interfacial binding sites were identified in this study.

Our studies identified two kinds of lipid binding site on mPPases, an interfacial binding site and a distal binding site. The residues at the interfacial study are not highly conserved, ranging from 47.8%– 61.8% conservation. This is low for functionally relevant residues, but there are the repeated interactions with residues around K9.7X, such as K9.70 (Tm-PPase) and K9.73 (Cp-PPase), around K/R13.52 (K in Tm-PPase and Vr-PPase (K13.48) and R in Cp-PPase) and residues around K/R14.48 (K14.48 in Tm-PPase, K14.48 in Vr-PPase and R14.48 in Cp-PPase). These differ in position by one helix turn, and so face out into the membrane, thereby preserving their function and potentially accommodating the change in headgroup size between POPA and PIP 2 . In addition to conservation of interactions with specific residues, they are positioned on functionally relevant helices. This clustering of interactions on TMH that form the dimer interface and are linked to inter-subunit communication and K+-dependency clearly supports our hypothesis that presence of anionic lipids at the interfacial interaction site may be functionally relevant to the mPPase catalytic cycle.

In addition to the computational evidence laid out in this work, there is experimental evidence of binding at the interfacial site. In the highest resolution mPPase structure currently available (Vr-PPase (PDB: 4A01)) [7], n-decyl-β-D-maltopyranoside (DM) detergent molecules are situated in this proposed binding site. In particular, the binding of one of these is highly similar to the anionic lipid positioning seen in our CG and AT simulations. Moreover, hydrogen bonds form between this detergent molecule and the K9.70 and K10.49 residues, mirroring the Tm-PPase simulations. The structural evidence of binding at the interfacial site in a mPPase provides further support to this region acting as a lipid binding site. The replacement of lipid with detergent was likely due to the purification and crystallisation conditions promoting removal of even tightly bound lipids [41]. However, the binding of this detergent may have acted similarly to a lipid and helped maintain oligomeric stability, particularly as it has been suggested that detergents can bind in lipid interaction sites [41].

The interacting residues at the distal interaction site are not highly conserved (20.8%– 64%), but interactions cluster at similar structural positions. In all proteins in this study, interactions between anionic lipids and the proteins were formed with TMH 1, 2, 3 and 4, with R/K1.60 (R in Tm-PPase, and K in Vr-PPase Cp-PPase). Positively charged residues within one helix turn from R/K1.60, at K1.61 (Tm-PPase), K1.62 (Vr-PPase) and R1.56 (Cp-PPase) and residues in the midpoints of the helices: K3.59, R3.63 and R4.48 (Tm-PPase), K2.50, K3.59 and K4.44 (Vr-PPase), and K2.46, R2.47 and K3.59 (Cp-PPase) also formed large number of interactions with anionic lipids. In previous simulations of Tm-PPase, the distal region of the protein has been found to be flexible [42], and does not have high conservation between species. Therefore, our simulations suggest that the interactions are conserved to specific helix turns rather than individual residues. This might explain why despite the lower sequence similarity, we observe similar interactions with anionic lipids in the distal area. The distal lipid interaction sites may play a role in mPPase stability, as the region was identified during a case study of the IMPROvER (integral membrane protein stability selector) pipeline for stabilising mutations [43]. Further, three of the Cp-PPase residues identified by IMPROvER were found to interact with anionic lipids in our study (F1.53, R3.67 and R7.62). We were unable to study effects of lipid interaction on protein stability, as in the CG simulations the elastic force network prevents the protein structure from substantially changing, although the loops were able to move. Conversion to all-atom systems partly overcame this, as the elastic force network was removed. Despite this, due to the corroborative results between this study and the IMPROvER study, the distal lipid interaction sites found in this work may play a role in protein stability.

Our study also provides evidence that lipid interactions in the distal and interfacial region may be a more general property of mPPase subfamilies, as Tm-PPase is a K+-dependent Na+-PPase, Vr-PPase is a K+-dependent H+-PPase and Cp-PPase is a K+-dependent Na+/H+-PPase. Despite the conserved pattern of interactions in our studies, there was no conserved sequence motif to identify these interactions in other family members, so likely homology modelling and electrostatic profile analysis, as performed here for Cp-PPase, would be required to identify binding sites in other homologues. The retention of interactions between pumping specificities is perhaps not unexpected, as the residues involved in coordinating the pumped ion are at the centre of the mPPase structure [1]. However, the conservation across subfamilies of lipid interactions that may stabilise the protein and be of mechanistic relevance bodes well for future structural and functional research using alternative homologues.

The preferential interaction of Vr-PPase with PIP 2 in this work was striking, as PIP lipids are known to have roles in signalling and protein-protein interactions and are often found bound to proteins of interest [44]. The function of Vr-PPase in the tonoplast membrane of plants has been linked to auxin regulation and signalling [45] and cooperative function with soluble pyrophosphatases [46]; this, taken with the evidence of PIP 2 binding in this study, may suggest a mechanism by which this signalling is able to take place. Additionally, the roles of the vacuolar ATPase complex and Vr-PPase are closely aligned [1], which may also be mediated by PIP 2 binding and activity.

This work provides the first evidence that interactions can form between mPPases and anionic lipids and that these are in regions that may hold functional significance and are conserved across homologues. These observations are very promising for future mPPase research. The role of the distal interaction site in stability needs to be investigated further, but co-crystallisation with lipids or mutagenesis to stabilise this region in detergent may be promising for structural studies and characterisation of other mPPases. Additionally, the putative role of the interfacial site in inter-subunit communication and catalysis warrants further investigation as it may be another factor in the apparent increasing complexity of the mPPase catalytic cycle.

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