Dimyristoylphosphatidylglycerol

DMPC (Dimyristoylphosphatidylcholine) and DMPG (Dimyristoylphosphatidylglycerol) were purchased from Avanti (Avanti Polar Lipids, AL, USA). Peptides are identified by their publication names, by their UniProtKB or genebank (gb|) accession number followed by the indication of the first and last aminoacid residues in brackets. Penetratin, B4FGE3(22–37), A3KLW0(117–136), Q7YRI0(9–28) were purchased from ImmunoKontact (AMS Biotechnology, UK); gb|ACU24018.1|(73–101), gb|AAD22970.1|(120–148) and Q9XEY7(120–148) were purchased from JPT Peptide Technologies GmbH, Germany. The IAPs Q8KG25(327–351), P94692(929–955), P61458(35–60), B0CZJ3(104–130), A4HW34(187–217), Q8RW88(70–95), Q6TV81(25–52), O43312(33–62), A5LDU0(184–211), P83637(1–12) as well as DS 01 (P83637), DS 01(1–12), Nattererin-1 (P86913), Syphaxin (P85279), Phes (HQ012497) HSP-4 (JF916646), Pseudin B (P86915), PS-2 (P84567), Magainin-2 amide (P11006), Hyposin HA-6 (P86921) were synthesized in-house by solid-phase chemistry. Peptides were purified, mass analyzed using an Ultraflex III (Bruker Daltonics, Germany) and quantified by their corresponding molar absorptivities or by the method of Wadell [47] (link).

Total lipid extracts were analyzed by hydrophilic interaction liquid chromatography using a high-performance liquid chromatography (HPLC) Ultimate 3000 Dionex (Thermo Fisher Scientific, Bremen, Germany) with an autosampler coupled online to a Q-Exactive hybrid quadrupole mass spectrometer (Thermo Fisher, Scientific, Bremen, Germany). The solvent system and instrumental settings were set as described previously [28 (link)]. To perform the HILIC-LC-MS analyses, 10 µg of total lipid extract, 2 µL of phospholipid standards mix (0.01 µg dimyristoylphosphatidylcholine (dMPC), 0.01 µg dimyristoylphosphatidylethanolamine (dMPE), lysophosphatidylcholine (LPC) 0.01 µg, 0.04 µg dipalmitoylphosphatidylinositol (dPPI), 0.006 µg dimyristoylphosphatidylglycerol (dMPG), 0.02 µg dimyristoylphosphatidylserine (dMPS), 0.04 µg tetramyristoylcardiolipin (tMCL), 0.01 µg sphingomyelin (SM(17:0/d18:1)), 0.04 µg dimyristoylphosphatidic acid (dMPA)) and 88 µL of eluent (40% of mobile phase A and 60% of mobile phase B) were mixed and injected into the Ascentis Si column HPLC Pore column (15 cm × 1 mm, 3 µm, Sigma-Aldrich, St Louis, USA), with a flow rate of 40 µL minutes⁻¹ at 30 °C. Acquisition in the Orbitrap® mass spectrometer was performed in both positive (electrospray voltage 3.0 kV, Thermo Scientific, Waltham, USA) and negative (electrospray voltage −2.7 kV) modes. For lipidomic analysis, phospholipid peak integration and assignments were performed using MZmine version 2.32 (Boston, USA) [29 (link)]. For all assignments, ions within 5 ppm of the lipid exact mass were considered. Analysis of the MS/MS spectra acquired in the positive ion mode was performed to confirm the identity of the molecular species belonging to the MGMG, DGMG, MGDG, DGDG, DGTS, PC and LPC classes. The MS/MS spectra acquired in the negative ion mode were used to confirm the identity of SQDG, SQMG, LPE, PE, LPG, PG, LPI, PI, lysophosphatidic acids (LPA) and phosphatidic acids (PA). Negative ion mode MS/MS data were used to identify the fatty acid carboxylate anion fragments RCOO⁻, which allowed the assignment of the fatty acyl chains esterified to the PL precursor. All the ions detected and MS/MS fragmentation patterns characteristic of the lipid classes detected and analyzed in the present study, acquired both in positive and negative ion modes, are available online as Supplementary Information (Supplementary Table S1 and Supplementary Figure S1). Normalization of the data was performed by dividing the peak areas of the extracted ion chromatograms (XICs) of the polar lipid precursors of each class (listed in Supplementary Table S1) by the peak area of the internal standard selected for the class.

Liposomes decorated with two antigens, mCD52 (peptide with the amino acid sequence TSSPSAD, which is a CD52 mimotope; synthesized by Aimee Boyle, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands) and DNP, encapsulating sulforhodamine B (20 mM; Sigma-Aldrich, S1402) in PBS were produced. Lipid films were composed of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), cholesterol, DNP-cap-PE and mCD52-cholesterol (44:5:49:1:1 mol%). Liposomes were prepared as described previously (24 (link)–26 (link)). All lipids, except mCD52-cholestrol, were purchased from Avanti Polar Lipids.

We constructed models of the channel based on the crystal structure with code 3UKM, which was prepared at a pH >7.4 and is believed to represent an open conformation. These models were protonated to simulate either pH6.0 or pH7.4 and were embedded in a double bilayer system, following a previously established method^{11 (link)}. It’s important to note that this system was different from the one used for the pKas calculations. In each extracellular-facing leaflet of the bilayer, there were 105 molecules of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC) and 10 of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE). To partially recreate the negative charge inside biological membranes, the cytoplasmic-facing leaflet contained 10 negatively charged dimyristoylphosphatidylglycerol (DMPG) molecules and 105 POPC molecules only. We show in SI-2 section that this membrane modification is unlikely to significantly affect the pKas of the membrane inserted protein. The numbers of lipids were chosen to ensure that both leaflets had a comparable surface area, considering the structure of the channel. After solubilisation in 150 mmol $\cdot$ L-1 KCl and neutralisation, a typical simulation box contained $~$ 243’000 atoms. For the wild-type (WT) sequence, we performed a total of 18 trajectories at each pH, resulting in a cumulative simulation time of 5.8 μs at pH 7.4 and 4.8 μs at pH6.0. A similar procedure was followed for the H122N mutant, except that in this case, we conducted four simulations, each lasting 300 ns, at each pH value. Therefore, the total trajectory time dedicated to the H122N mutant was 2.4 μs. The all-atom MD simulations were performed with the CHARMM36 force field^{31 (link)} using the GROMACS package version 2021.5^{24 (link)}. The TIP3P water model was used^{28 (link)}. Bond and angle lengths involving hydrogen atoms were constrained using the LINCS algorithm allowing an integration time step of 2 fs. Short-range electrostatics were cut off at 1.2 nm. Van der Waals interactions were calculated explicitly up to 10 Å, beyond which a switch function was used to smoothly switch off the interactions to reach zero at 12 Å. Long-range electrostatic interactions were calculated by the PME algorithm^{32 (link)}. The protein, lipids, and water/ions were coupled separately to a temperature bath at 310 K with the Nose-Hoover method with a time constant of 1.0 ps^{33 (link),34 (link)}. The system pressure was kept constant by semi-isotropic Parrinello-Rahman coupling to a reference value of 1 bar as implemented in the GROMACS suite. GROMACS and in-house Python scripts were used to analyse the data. Time series analyses reveal that the Cα RMSD approached values close to 3 Å over the course of the simulations, but without achieving prefect convergence. More precisely, the interactions studied in this article converged significantly and rapidly, enabling us to incorporate all values after 50 ns of stabilisation in our calculations. Some distances involving contact losses required more time to converge (Fig. S1). To determine the orientation of residue 122 with respect to the main axis of the channel, the structures were aligned along their main axis. The vertical red arrow in Fig. 4C shows this orientation. A second vector links the Cα atom of residue 122 with the center of mass of the selected side chain pairs, which are ND1 and NE2 in the case of histidine and OD1 and ND2 in the case of asparagine. The angle between these two vectors determines the orientation of the residue.