Relevance of the protein macrodipole in the membrane-binding process. Interactions of fatty-acid binding proteins with cationic lipid membranes

Abstract

The fatty acid-binding proteins L-BABP and Rep1-NCXSQ bind to anionic lipid membranes by electrostatic interactions. According to Molecular Dynamics (MD) simulations, the interaction of the protein macrodipole with the membrane electric field is a driving force for protein binding and orientation in the interface. To further explore this hypothesis, we studied the interactions of these proteins with cationic lipid membranes. As in the case of anionic lipid membranes, we found that both proteins, carrying a negative as well as a positive net charge, were bound to the positively charged membrane. Their major axis, those connecting the bottom of the β-barrel with the α-helix portal domain, were rotated about 180 degrees as compared with their orientations in the anionic lipid membranes. Fourier transform infrared (FTIR) spectroscopy of the proteins showed that the positively charged membranes were also able to induce conformational changes with a reduction of the β-strand proportion and an increase in α-helix secondary structure. Fatty acid-binding proteins (FABPs) are involved in several cell processes, such as maintaining lipid homeostasis in cells. They transport hydrophobic molecules in aqueous medium and deliver them into lipid membranes. Therefore, the interfacial orientation and conformation, both shown herein to be electrostatically determined, have a strong correlation with the specific mechanism by which each particular FABP exerts its biological function.

Fig 1. Charge distribution of ReP-NCXSQ and L-BABP.

Cartoon representation of L-BABP (panel A) and ReP1-NCXSQ (panel B). Macrodipoles are represented as arrows. The macrodipole vector of L-BABP is scaled 2.5 times greater than ReP1-NCXSQ to facilitate visualization. The electrostatic potential is represented in transparent surface; the isosurfaces were set in +1 mV (blue) and -1 mV (red).

Fig 2. Initial and final configurations of the simulations of L-BABP.

Initial (upper panels) and final (lower panels) configurations of the 100 ns simulations of L-BABP with EDMPC at pH 6.8, 10 mM NaCl. B-EDMPC1 (panels A and C) and B-EDMPC2 (panels B and D). The arrows represent the macrodipoles. N atoms in choline and O atoms in phosphate groups are in blue and red respectively.

Fig 3. Initial and final configurations of the simulations of ReP1-NCXSQ.

Initial (upper panels) and final (lower panels) configurations of ReP1-NCXSQ in EDMPC at pH = 6.8, 10 mM NaCl. R-EDMPC1 (panels A and E), R-EDMPC2 (panels B and F), R-EDMPC3 (panels C and G) and R-EDMPC4 (panels D and H). The arrows represent the macrodipoles. N atoms in choline and O atoms in phosphate groups are in blue and red respectively.

Fig 4. Trajectories of the molecular dynamics simulations of adsorption.

The z_L-P (panel A and B), the θ_{μ-plane x-y} (panel C and D) and the root mean square deviation (RMSD) (panel E and F) for the four simulations of ReP1-NCXSQ with cationic membranes of EDMPC (R-EDMPC1-4) and the two of L-BABP with EDMPC (B-EDMPC1-2) in panels A, C and E. In order to have a reference, data from simulations of ReP1-NCXSQ with anionic membranes of POPG (R-POPG1-3) [8] are displayed in panels B, D and F.

Fig 5. Histograms of the “bound” state over the last 70 ns of the molecular dynamics simulations of the adsorption processes.

z_L-P (panel A) and θ_{μ-plane x-y} (panel B) for the four simulations of ReP1-NCXSQ with cationic membranes of EDMPC, the two of L-BABP with EDMPC, and of ReP1-NCXSQ with anionic membranes of POPG [8]. Colour coding are as in Fig 4.

Fig 6. FTIR spectra of ReP1-NCXSQ.

Spectra in solution (SOL) and in the presence of anionic membranes of DMPG and cationic membranes of DMTAP and EDMPC in this order from top to bottom. Samples were at 33 ^oC. Measured spectra: lower black traces. Fourier self-deconvolutions: upper gray traces, band width = 18 cm^-1 and factor k = 2. For reference, dotted lines were traced at 1625, 1630 and 1650 cm^-1.

Fig 7. Component analysis of the FTIR spectra.

ReP1-NCXSQ in solution (panel A) and in the presence of anionic membranes of DMPG (panel B) and cationic membranes of DMTAP (panel C) and EDMPC (panel D). Spectra were collected a 33 ^oC. Measured spectra (lower continuous black trace), Fourier self-deconvolutions (upper continuous gray trace), using bandwidth of 18 cm^-1 and factor k = 2. Band components are in dashed lines.

Fig 8. Cartoon representation of several human FABPs with their macrodipole.

Macrodipoles (μ) are represented as arrows. The macrodipole was calculated according to GROMOS96 partial charges. A: Heart FABP (PDB ID 1G5W); μ = 158 D. B: Intestine FABP (PDB ID 1KZW); μ = 199 D. C: Adipocyte FABP (PDB ID 3FR4); μ = 361 D. D: Epidermal FABP (PDB ID 1JJJ); μ = 272 D. E: Liver FABP (PDB ID 2F73); μ = 151 D. Moment dipole vector scaling is not linear with the dipole moment magnitude.