Ladderane phospholipids form a densely packed membrane with normal hydrazine and anomalously low proton/hydroxide permeability
Contributed by Steven G. Boxer, July 30, 2018 (sent for review June 24, 2018; reviewed by Erick Carreira and Marjorie Longo)
Significance
Ladderane lipids represent exotic natural products containing highly strained, concatenated cyclobutane rings, a motif that has not been found in any other natural products. These lipids are exclusively found in bacteria that carry out anaerobic ammonium oxidation (anammox), suggesting a biological role in the anammox process. The relationship between molecular structure and this metabolism remains unexplored due to a lack of natural lipid material to study. We use an efficient chemical synthesis to create large quantities of pure ladderane lipids for biophysical analysis. This analysis reveals some unusual properties of membranes composed of ladderane lipids. Significantly, ladderane membranes have low proton permeability, which would slow the breakdown of the proton gradient used to synthesize ATP during the slow anammox metabolism.
Abstract
Ladderane lipids are unique to anaerobic ammonium-oxidizing (anammox) bacteria and are enriched in the membrane of the anammoxosome, an organelle thought to compartmentalize the anammox process, which involves the toxic intermediate hydrazine (N2H4). Due to the slow growth rate of anammox bacteria and difficulty of isolating pure ladderane lipids, experimental evidence of the biological function of ladderanes is lacking. We have synthesized two natural and one unnatural ladderane phosphatidylcholine lipids and compared their thermotropic properties in self-assembled bilayers to distinguish between [3]- and [5]-ladderane function. We developed a hydrazine transmembrane diffusion assay using a water-soluble derivative of a hydrazine sensor and determined that ladderane membranes are as permeable to hydrazine as straight-chain lipid bilayers. However, pH equilibration across ladderane membranes occurs 5–10 times more slowly than across straight-chain lipid membranes. Langmuir monolayer analysis and the rates of fluorescence recovery after photobleaching suggest that dense ladderane packing may preclude formation of proton/hydroxide-conducting water wires. These data support the hypothesis that ladderanes prevent the breakdown of the proton motive force rather than blocking hydrazine transmembrane diffusion in anammox bacteria.
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Lipid membranes are universal features of living systems. Along with membrane proteins, they form the internal and peripheral barriers of cells and organelles and maintain nonequilibrium states necessary for life. Cells produce a diverse array of lipid structures and expend considerable energy to tightly control the lipid compositions of their membranes (1). In conventional phospholipid bilayers, it is known that longer hydrocarbon tails and fewer degrees of unsaturation reduce fluidity and slow lateral diffusion, as well as affecting phase behavior (Fig. 1A) (2, 3). However, the physical properties and biological functions of lipids with unconventional structures (SI Appendix, Fig. S1) are mostly unexplored.
Fig. 1.
The ladderane lipids, containing lipid tails terminating in either a [3]-ladderane motif (highlighted in blue in Fig. 1B) or a [5]-ladderane motif (highlighted in red in Fig. 1B), occur uniquely in anaerobic ammonium-oxidizing (anammox) bacteria and are some of the most structurally exotic lipids known (4–9). Anammox bacteria are not available as a pure culture, and enrichment cultures have doubling times of 1–2 wk (10, 11). Isolation of lipids from anammox enrichment cultures yields a complex and inseparable mixture, preventing biophysical characterization of individual ladderane species (12, 13). To enable experiments on ladderane lipids, we developed de novo enantioselective chemical syntheses of both [3]- and [5]-ladderane lipid tails and ladderane phospholipids (14). We prepared a [5]-ladderane–[3]-ladderane phosphatidylcholine ([5][3]PC; Fig. 1B) and a di-[3]-ladderane phosphatidylcholine ([3][3]PC; Fig. 1B), two of the most common ladderane phospholipids across a range of anammox genera (8). Additionally, we prepared di-[5]-ladderane phosphatidylcholine ([5][5]PC; Fig. 1B), which is not known to occur naturally.
Ladderanes are enriched in the membrane of the anammoxosome, a specialized organelle within which anammox catabolism is thought to occur (4). Ammonium and nitrite are coupled to produce dinitrogen via intermediate hydrazine (N2H4); oxidation of hydrazine to dinitrogen is highly exergonic, and this free energy is believed to be harnessed to pump protons across the anammoxosome membrane (15–17). The resulting pH gradient of ∼1 unit in turn powers ATP synthesis (Fig. 1C) (18–20). Hydrazine’s cellular toxicity and bioenergetic value as a metabolic intermediate have led to the hypothesis that ladderanes serve to prevent the diffusion of hydrazine out of the anammoxosome, thereby protecting the contents of the riboplasm and periplasm from free hydrazine while preserving metabolic energy (4, 20). It has also been reasoned, based on long anammox doubling times and slow hydrazine synthase activity, that ladderanes might prevent the passive diffusion of protons out of the anammoxosome to preserve the proton motive force necessary for ATP synthesis (4, 21). Another theoretical study suggests ladderanes might trap reactive species such as free radicals in addition to protons (22). Although studies on enrichment cultures and lipid extracts suggest that the anammoxosome membrane is dense and relatively impermeable to dyes, hypotheses about hydrazine and proton permeability remain to be experimentally tested (4, 12). We recently completed the total synthesis of a ladderane phospholipid, which we expand in this work to a total of three phospholipids with distinct tail structures for structure–function studies (14). Biophysical experiments on model membranes composed of these synthetic lipids allow us to identify properties of individual molecular ladderane species.
Results
Syntheses of Ladderane PCs.
We recently reported enantioselective total syntheses of a [5]-ladderane fatty acid and a [3]-ladderane fatty alcohol (14). These lipid tails were then elaborated to [5][3]PC, [3][3]PC, and [5][5]PC using a modification of our previously reported phosphatidylcholine synthetic route (SI Appendix, Schemes S1–S3).
Differential Scanning Calorimetry.
Lipid films were hydrated with a low-melting and high-boiling 1:1 ethylene glycol/phosphate buffer mixture, and differential scanning calorimetry (DSC) was performed to measure the transition temperature (Tm) of the multilamellar lipid dispersions. Control experiments showed that the effect of ethylene glycol on Tm is negligible (SI Appendix, Table S1). A single transition between −40 and 80 °C was observed for each ladderane PC. The presence of a [3]-ladderane tail at the sn-2 position apparently ensures a low Tm (compare [5][3]PC, Tm = 11.8 °C to [5][5]PC, Tm = 68.4 °C; Fig. 2A). We believe the cyclohexane ring in the [3]-ladderane motif introduces a kink in the lipid tail, mirroring the fluidizing effect of cis-unsaturation in the sn-2 position tail of a straight-chain lipid [compare 18:0–18:1 PC (SOPC), Tm = 4.1 °C to di18:0 PC (DSPC), Tm = 57.1 °C]; [5][5]PC has approximately the same Tm as di20:0 PC (DAPC).
Fig. 2.
Self-Assembly of Ladderane Phospholipids into Lipid Bilayers.
We next evaluated whether ladderane PCs would self- assemble into giant unilamellar vesicles (GUVs) using established methods (23–25). Upon gentle hydration, films of [3][3]PC and [5][3]PC appeared to form GUVs (Fig. 2 B and C) with homogeneous incorporation of dye [0.1 mol % Texas Red-DHPE (TR-DHPE)] and spherical shapes confirming that these lipids form fluid bilayers at room temperature. Formation of GUVs from [5][5]PC required heating above its Tm, and upon cooling we observed fluorescent objects similar to gel-phase GUVs formed from DAPC (Fig. 2D and SI Appendix, Fig. S3 A and B). A 1:1 mixture of [3][3]PC and [5][5]PC formed GUVs with visible domains that exclude TR-DHPE (Fig. 2E) in a manner analogous to mixtures of straight-chain phospholipids that form coexisting phases at room temperature (26). Hydration of [3][3]PC or [5][3]PC films followed by extrusion through a 50-nm-pore polycarbonate membrane resulted in the formation of small unilamellar vesicles (SUVs) (SI Appendix, Table S3).
Hydrazine Transmembrane Diffusion Assay.
To test the hypothesis that ladderanes form a barrier to transmembrane diffusion of hydrazine, we developed an assay based on a small-molecule sensor that fluoresces upon condensation with hydrazine (27). We prepared a derivative with added hydrophilic sulfonate and amide groups to improve aqueous solubility and presumably reduce interactions between the dye and the membrane (SI Appendix, Figs. S4–S11 and Schemes S4–S6); the final hydrazine sensor (HS)-based hydrazine transmembrane diffusion kinetic assay design is illustrated in Fig. 3A (28). We also confirmed that HS does not localize in lipid bilayers (SI Appendix, section 9).
Fig. 3.
We performed this hydrazine diffusion assay on SUVs composed of several straight-chain PCs, [3][3]PC, and [5][3]PC. SUVs encapsulating HS in a pH = 7.4 phosphate buffer were added to an equiosmolar pH = 7.4 buffer containing hydrazine, and changes in the fluorescence intensity were measured (Fig. 3B). Among straight-chain PCs, hydrazine transmembrane diffusion rates depended strongly on hydrophobic tail length (Fig. 3C), likely due to different bilayer thicknesses among these lipids (29–31). SUVs of ladderane PCs exhibited half-lives (t1/2s) of hydrazine transmembrane diffusion well within the range exhibited by SUVs of straight-chain PCs. We have also conducted this diffusion assay at the anammoxosome-relevant pH of 6.3 with SOPC, di22:1 PC (DEPC), and [3][3]PC. Transmembrane diffusion rates are all slower, as expected, but maintain the same trend as at pH = 7.4 (SI Appendix, section 10).
Small-Angle X-Ray Scattering.
To examine the relationship between hydrazine diffusion rate and bilayer thickness, we performed small angle X-ray scattering (SAXS) on rigorously extruded SUVs of unsaturated straight-chain PCs and ladderane PCs (Fig. 3C). Bilayers of straight-chain PCs increased in thickness with increasing chain length from 3.25 nm for di16:1 PC to 4.37 nm for di22:1 PC. The [3]-ladderane and [5]-ladderane tails in our synthetic ladderane phospholipids each contain 20 carbons, and [3][3]PC and [5][3]PC formed bilayers of about the same thickness as di20:1 PC (∼4 nm). SAXS curves also confirmed the high level of unilamellarity of SUVs (see discussion in SI Appendix, section 12).
pH Equilibration Across Membranes.
To test the hypothesis that ladderanes form a barrier to transmembrane diffusion of protons/hydroxide ions, we performed a carboxyfluorescein (CF)-based assay of pH equilibration (Fig. 4A) on SUVs of several straight-chain PCs, [3][3]PC, and [5][3]PC (32). SUVs encapsulating CF at pH = 7.2 in bis-Tris propane buffer were added to equiosmolar pH = 5.8 bis-Tris propane buffer, creating a transmembrane pH gradient that spontaneously decayed over time. The decrease in CF fluorescence was used to monitor the decrease in pH inside the SUVs as protons/hydroxide ions equilibrated across the bilayers. We also performed control experiments with valinomycin to confirm that a buildup of membrane potential was not affecting relative pH equilibration rates and with gramicidin to confirm that vesicles were unilamellar. We confirmed that valinomycin inserts into ladderane bilayers with the fluorescent K+ sensor PBFI (SI Appendix, section 13) (33). Equilibration of pH across [3][3]PC and [5][3]PC bilayers was approximately an order of magnitude slower than across membranes composed of straight-chain PCs (48–75 min vs. 0.33–6.9 min t1/2) (Fig. 4 B and C). We observe a correlation between Tm and pH equilibration t1/2 for straight-chain PCs; however, [5][3]PC and [3][3]PC deviate from this trend, suggesting that unique intermolecular interactions between ladderane lipid tails affect rates of pH equilibration (Fig. 4D). These interactions remain present in a mixture of [3][3]PC with 16:0–18:1 PC (POPC) as indicated by a pH equilibration t1/2 of 31 min (SI Appendix, section 13).
Fig. 4.
Pressure-Area Isotherms of Langmuir Monolayers.
To help explain the low proton/hydroxide permeability of ladderane PC bilayers, we investigated the physical properties of PC monolayers at the air–water interface. Ladderane PC monolayers collapse at surface pressures similar to liquid-phase monolayers of POPC (Fig. 5A), yet their compressibilities, C, are similar to those of solid-phase monolayers of DAPC (Fig. 5C). In addition, monolayers of ladderane phospholipids have mean molecular areas (MMAs) smaller than fluid straight-chain PCs (Fig. 5C). These data suggest that at room temperature the ladderane PC monolayers exist in a phase that is fluid but more tightly packed than straight-chain PCs. Pressure-area isotherms for [5][3]PC and [3][3]PC are qualitatively similar to the previously published isotherm for a ladderane PC extract (12).
Fig. 5.
Fluorescence Recovery After Photobleaching.
To quantitate fluidity in ladderane PC bilayers, we performed fluorescence recovery after photobleaching (FRAP) experiments on glass-supported lipid bilayers (SLBs) using Oregon Green-DHPE (OG-DHPE) as a fluorescent probe (Fig. 5B). As expected for lipids in a gel phase at room temperature, SLBs of [5][5]PC showed no lateral diffusion (Fig. 5B). SLBs of [5][3]PC and [3][3]PC exhibited lateral diffusion coefficients, D, at least one order of magnitude lower than SLBs of straight-chain PCs (0.24–0.29 µm2/s vs. 2.73–3.93 µm2/s) (Fig. 5 B and C). Straight-chain PCs show a linear correlation between D and Tm while ladderane PCs deviate from this trend (Fig. 5D).
Discussion and Conclusions
The facile preparation of vesicles, monolayers, and supported bilayers of [3][3]PC and [5][3]PC was not anticipated, as their unique structural elements might be expected to alter their molecular spontaneous curvature and favor nonbilayer structures. However, self-assembly is consistent with the observation that ladderanes are the primary lipid components of the membranes in anammox bacteria, suggesting a major structural role for ladderane phospholipids. Anammox bacteria rely on a pH gradient to drive ATP synthesis (19, 20). Loss of this pH gradient during the slow anammox metabolism would make this process unviable as a source of energy for the cell. Our data support the hypothesis that ladderanes evolved at least in part to impede the loss of this vital pH gradient. Our biophysical results are qualitatively similar to results from complex mixtures of ladderanes, but we are able to observe functional distinctions between individual molecules that are lost in mixtures (for a discussion see SI Appendix, section 16) (12, 13). We note that this study was enabled by the success of a scalable natural product total synthesis (14).
Although much work has been done to understand the permeability of membranes to easily detectable drugs and organic dyes, methods of quantifying transmembrane diffusion of small hydrophilic molecules such as hydrazine in real time are limited, often to indirect assays (34–36). The lack of correlation between the rates of pH equilibration and hydrazine transmembrane diffusion provides experimental evidence that these processes occur by different mechanisms. Evidence from the literature supports a model in which proton/hydroxide diffusion occurs via water molecules in the bilayer that act as proton wires or water clusters that carry protons (30, 37–40). Transmembrane diffusion of other ions (e.g., K+) and small neutral molecules (e.g., H2O, O2, and CO2), the rates of which are directly dependent on bilayer thickness, is thought to occur via direct partitioning into the bilayer and diffusion through the hydrophobic region of the bilayer (21, 41). Our data are consistent with the idea that this is the case for hydrazine. Our data do not support the hypothesis that ladderane PCs alone offer any advantage with respect to hydrazine permeability. Ladderane PC bilayers and straight-chain PC bilayers have similar rates of hydrazine transmembrane diffusion. Notably, the relative hydrazine permeability of ladderane bilayers compared with conventional PC bilayers was correctly predicted by molecular dynamics simulations (42). Hydrazine containment by other means, such as rapid conversion within the hydrazine synthase complex or retention within encapsulin nanocompartments, remains a possibility (17, 43).
In contrast, equilibration of pH across the [3][3]PC bilayer was at least 10 times slower than across bilayers composed of any straight-chain PC. To the best of our knowledge this is the slowest known pH equilibration to be measured across a homogeneous PC bilayer; interestingly, very slow pH equilibration across archaea-inspired tetraether lipid monolayers is also known (33). Rates of pH equilibration for straight-chain PC bilayers correlate with their Tms, which reflect the strength of intermolecular interactions and lipid packing. Our data are consistent with a model in which water must disrupt lipid–lipid interactions to form a proton-conductive wire or cluster in the hydrophobic region of the bilayer. For straight-chain PCs these interactions are accurately reflected in the Tms, but this correlation breaks down with [3][3]PC and [5][3]PC. Monolayers of [3][3]PC and [5][3]PC at room temperature have smaller MMAs and lower compressibilities than fluid straight-chain PC monolayers, suggesting tighter packing and a higher level of molecular order in the fluid bilayer that is somehow not reflected in the Tm. However, these stronger intermolecular interactions are reflected in the slower lateral diffusion rates in SLBs. Finally, it is interesting to note that [3][3]PC and [5][3]PC, which both have 20-carbon ladderane tails, form bilayers of similar thickness to di20:1 PC, indicating that the conformational rigidity of the polycyclobutane ladderane motifs counterbalances their shortened structure for a null net effect on bilayer thickness.
Strong London dispersion interactions between ladderane hydrocarbons have been predicted computationally (44). The strong ladderane–ladderane interactions might resist the formation of a proton-conductive water cluster/wire, which would disrupt these interactions (35, 39, 41, 42). This is consistent with the very low D of ladderane bilayers, as this same disruption of packing is necessary for the OG-DHPE to diffuse laterally. D correlates well with pH equilibration t1/2 for all PCs assessed, including ladderanes, indicating that D reports on the strength of the physical interactions relevant to proton/hydroxide permeability (SI Appendix, Fig. S22). Hydrazine, which is much smaller than a water cluster, may be able to diffuse through small, transient spaces without wholly disrupting ladderane–ladderane packing (12, 45). More generally, we have demonstrated that lipid packing in the hydrophobic region of the lipid bilayer can strongly affect the permeability of the bilayer to small ions. Cells may utilize this mechanism to control the permeabilities of their membranes.
Acknowledgments
We thank Dr. Stephen R. Lynch for assistance with NMR spectroscopy and Katherine N. Liu for assistance in the development of the hydrazine transmembrane diffusion assay. This work was supported by Stanford University, the Terman Foundation, National Institutes of Health Grants GM069630 and GM118044 (to S.G.B.), the NSF Biophysics Program (S.G.B.), the National Science Foundation (graduate fellowships to J.A.M.M., C.M.C., and S.R.S.), and the Center for Molecular Analysis and Design (graduate fellowship to F.R.M.). Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Part of this work was performed at the Stanford Nano Shared Facilities, supported by the NSF under Award ECCS-1542152.
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Cover image: Pictured are cardiac fibroblasts under simulated ischemic conditions. Svenja Hinrichs et al. report that treating heart cells with proadrenomedullin (ProADM), the putatively inactive protein precursor of the hormone adrenomedullin (ADM), increased cell survival during ischemia. ProADM exhibited proinflammatory effects in cardiac fibroblasts, unlike ADM, but antiinflammatory effects in activated immune cells. The results suggest that ProADM is biologically active and prevents cell death during heart attacks by regulating inflammation in conjunction with ADM. See the article by Hinrichs et al. on pages E8727–E8736. Image courtesy of Svenja Hinrichs.
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Published online: August 27, 2018
Published in issue: September 11, 2018
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Acknowledgments
We thank Dr. Stephen R. Lynch for assistance with NMR spectroscopy and Katherine N. Liu for assistance in the development of the hydrazine transmembrane diffusion assay. This work was supported by Stanford University, the Terman Foundation, National Institutes of Health Grants GM069630 and GM118044 (to S.G.B.), the NSF Biophysics Program (S.G.B.), the National Science Foundation (graduate fellowships to J.A.M.M., C.M.C., and S.R.S.), and the Center for Molecular Analysis and Design (graduate fellowship to F.R.M.). Use of the Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under Contract DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. Part of this work was performed at the Stanford Nano Shared Facilities, supported by the NSF under Award ECCS-1542152.
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The authors declare no conflict of interest.
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