The lipid mediator lysophosphatidic acid induces folding of disordered peptides with basic amphipathic character into rare conformations

Abstract

Membrane-active, basic amphipathic peptides represent a class of biomolecules with diverse functions. Sequentially close protein segments also show similar behaviour in several ways. Here we investigated the effect of the lipid mediator lysophosphatidic acid (LPA) on the conformation of structurally disordered peptides including extracellular antimicrobial peptides (AMPs), and calmodulin-binding motifs derived from cytosolic and membrane target proteins. The interaction with associated LPA resulted in gain of ordered secondary structure elements, which for most cases were previously uncharacteristic of the particular peptide. Results revealed mechanism of the LPA-peptide interactions with regulation of the lipid on peptide conformation and oligomerization in a concentration-dependent manner involving (1) relocation of tryptophan residues into the lipid cluster, (2) multiple contacts between the binding partners dictated by complex driving forces, (3) multiple peptide binding to LPA associates with an affinity in the low micromolar range, and (4) selectivity for LPA compared with structurally related lipids. In line with recent findings showing endogenous molecules inducing structural changes in AMPs, we propose that accumulation of LPA in signalling or pathological processes might modulate host-defense activity or trigger certain processes by direct interaction with cationic amphipathic peptide sequences.

Figure 1

Far-UV CD spectra of the peptides in the absence (black) and presence (red) of LPA. Spectra were collected with and without 100 μM LPA under low-salt conditions. The induced secondary structure is mainly helical for melittin (25 µM), mastoparan (25 µM) and peptide IP3R1 (36 µM) (top row), rich in β-sheet for eight peptides (GAP43IQ (36 µM), GAP43pIQ (36 µM), CM15 (24 µM), PMCA1 (21 µM), PMCA2 (26 µM), RYR (34 µM), Dhvar4 (18 µM), buforin (24 µM); middle rows), while no remarkable change was detected for IP3R2 (36 µM) and the control peptide (36 µM) (bottom row). Note that ellipticity scales are different.

Figure 2

Lipid-peptide interactions studied by tryptophan (Trp) fluorescence. Spectra were taken at peptide concentration of 3 μM with and without 100 μM LPA in high-salt buffer, and normalized pairwise to the maximal intensity (I_max) measured in the absence of the lipid. Note that each peptide alone showed emission maximum at 356 nm resulting in overlapping spectra which blue-shifted upon LPA addition as labelled in the figure, and listed in Table 2. Emission intensity variations at 1, 3, 6, and 10 μM are compared in Fig. S2.

Figure 3

LPA selectivity among the investigated peptide-lipid interactions. CD spectra for GAP43pIQ (36 µM) were measured in the presence and absence of lysophospholipids (lysophosphatidylcholine, LPC, sphingosylphosphorylcholine, SPC, and sphingosine, Sph) at 100 µM in high-salt buffer (a), and SDS at 100 μM in low-salt buffer (b), respectively. Only LPA affected peptide conformation.

Figure 4

Peptide interaction with liposomes containing LPA. CD spectra (a), and fluorescence emission spectra (b) for melittin (30 and 2 µM, respectively) in the presence of various liposomes. (c–f) CD spectra of peptides CM15 (36 µM), GAP43IQ (36 µM), buforin (36 µM), and PMCA2 (17 µM) in the presence of LPA-containing liposomes. All spectra were recorded in high-salt buffer. Spectra taken with LPA micelles (100 μM lipid) are also shown for comparison. For molar composition of the liposomes see Methods section. Nominal lipid concentrations for the liposomes were as follows: (a) PC, and PC/PG 1.3 mM, PC/Chol/PE and PC/Chol/PE/LPA 2 mM, (b) 100 μM for all liposomes. (c–f) PC/Chol/PE/LPA 1 mM.

Figure 5

Titration of IP3R1 with LPA (a) and SDS (b) exploiting tryptophan (Trp) fluorescence. Titration with LPA was performed in high-salt buffer while that with SDS in low-salt buffer, also used for CD spectroscopic experiments. Data were fitted to the Hill-equation, and yielded an apparent K_d of 19.0 ± 1.3 μM and a Hill-coefficient of 2.1 ± 0.36 for the IP3R1-LPA interaction. Values are mean ± SEM (n = 3).

Figure 6

Structural changes of peptide GAP43IQ induced by LPA and SDS traced by CD spectroscopy. (a–c). Spectra of the peptide recorded in the absence and in the presence of the lipids. (b–d) Lipid concentration-dependent changes in peptide conformation highlighting elements with pronounced alterations upon interaction. Secondary structure elements are according to the classification of the analysis method used considering three types of antiparallel β-sheet with different twists (cyan, blue and green). The content of all the individual β-forms, the total estimated β-conformation (black), and the disordered fraction (red) changed in the same lipid concentration range. Note that structural changes in the presence of SDS and LPA follow similar trends but take place at different concentrations, for LPA at CMC and for SDS much below the CMC.

Figure 7

Calorimetric traces for the LPA-peptide interaction. LPA (100 µM) was titrated with aliquots of the peptide GAP43IQ (200 μM) under high-salt (a) or low-salt (b) conditions. Points were fitted to the one set of sites model (solid lines), and parameters evaluated are listed in Table 3.

Figure 8

The effect of LPA on the peptide structure studied with ATR-FTIR. Complex formation was initiated by mixing the components with a peptide-to-lipid ratio of ~1:5 using concentrations of 200 μM and 1 mM for the peptide and the lipid, respectively. Spectra were collected for the mixture immediately after mounted (solution, a) as well as for the surface-dried sample (b), respectively.

Figure 9

Schematic model showing three possible arrangements for the investigated set of peptides upon interaction with LPA micelles. (a) A peptide in inserted helix conformation, (b) a peptide in helical conformation associating with the micelle, (c) a peptide in extended, sheet-like conformation. Note that in cases b and c, peptide hydrophobic side chains are most likely inserted into the more lipophilic area, beyond the headgroup region of the micelles. Location of hydrophilic residues is highlighted by dashed cyan fill on the corresponding side of the formed secondary structure. For the complete set of peptides and their side chain distribution upon forming helical or extended sheet-like conformation, see Fig. S4 in Supplementary Information.