Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding proteins

Abstract

Background: Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress.

Results: We performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2.

Conclusions: Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.

Keywords: Crystal structure; Fatty acid-binding protein; Membrane binding; Molecular dynamics; Mutation; Myelin; Protein stability.

Fig. 1

Analysis of P2 folding by CD spectroscopy. a Comparison of CD spectra of wt-P2, P2-F57A, and P2-P38G. The spectra for wt-P2 and P2-P38G were presented earlier [25]. b Spectra for the same P2 variants in the presence of lipids. The dashed lines indicate difference spectra between wt-P2 and each mutant. c Stability analysis of P2-F57A by CD in solution. CD spectra are shown from + 20 (black) to + 90 °C (blue), at intervals of 10 °C for clarity. The spectrum at + 50 °C is shown in red to aid in comparison between samples. d Stability of P2-F57A in the presence of lipids. Inset: CD signal at 200 nm of wt-P2 (black), F57A (red), and P38G (green) bound to lipids as a function of T

Fig. 2

Lipid binding experiments. a wt-P2 (black) and P2-F57A (red) initial vesicle aggregation profiles as a function of protein concentration. b Decrease in the turbidity signal over time in the mutant sample. c Co-sedimentation at 1:100 P/L (10 μM protein with 1 mM DMPC:DMPG) suggests roughly 50% binding of protein to vesicles. 1, molecular weight marker; 2, wt-P2 in pellet; 3, P2-F57A in pellet; 4, wt-P2 supernatant; 5, P2-F57A supernatant. d Co-sedimentation of wt-P2 with lipid vesicles. 1–2, supernatant and pellet of 20 μM P2 with 1 mM DMPC:DMPG; 3–4 supernatant and pellet of 20 μM P2 with 1 mM DOPC:DOPS. The asterisks indicate the positions of monomeric (*), dimeric (**), and trimeric (***) P2 in the proteolipid pellet. e DSC measurements show the diminished effect of the F57A mutant (red) on the lipid phase transition behaviour, while wt-P2 (black) has a clear effect. Dotted line: lipids alone, dashed line: P/L 1:200, solid line P/L 1:100. f Examples of SPR sensorgrams; shown are duplicate injections of 10 μM wt-P2 (black) and P2-F57A (red) onto immobilized DOPC:DOPS (1:1). g The steady-state affinity of the F57A mutants to DOPC:DOPS (1:1) vesicles is marginally weaker compared to wild type. All error bars are standard deviations

Fig. 3

Phe57 in the P2 crystal structure. a The location and interactions of Phe57 in wt-P2. Note the C-H...π bonds on both faces of the Phe57 side chain. b Conformational effects of the F57A mutation (stereo view). Superposed are wt-P2 (magenta) and all individual P2-F57A monomers from the three crystal forms (6 in total). Note the apparent flexibility of helix α2 and the β3-β4 loop. c Discontinuity of the β barrel of P2; a line of water molecules mediates contact between strands β4 and β5

Fig. 4

MD simulations of P2-F57A. a Fluctuation of the distance between Phe57 and helix α2 shows flipping of the Phe57 side chain in wt-P2 during the simulation. The simulation was run with (magenta) and without (black) the fatty acid ligand. b The two conformations of Phe57 (cyan). Left: Phe57 points inwards and interacts with the fatty acid (magenta). Right: Phe57 points outwards. Bovine P2 crystal structure is shown superposed in yellow, with the same conformation. c The structure shows the distance measured when studying barrel opening. d Distance between the tips of the β3-β4 and β5-β6 loops during the simulation. Black: wt-P2; red: F57A; green: P38G. Left: simulations without ligand. Right: simulations with bound palmitate. The red and green arrows indicate the positions of the snapshopt in the next panel. e Structural snapshots from the simulations. Left: Closed starting structure for F57A; middle: open F57A structure at 1.5 μs; right: open P38G structure with ligand at 2.2 μs, identified from our earlier trajectories [25]. f RMSF of wt-P2 (black) and F57A (red) in the simulations. Thick lines indicate unliganded simulations and thin lines those with bound fatty acid

Fig. 5

DCCM analysis of MD trajectories. a wt-P2 without fatty acid. Note the two regions of strong anti-correlation (arrows). b wt-P2 with palmitate. c P2-F57A without ligand. d P2-F57A bound to palmitate

Fig. 6

Flexibility and contact network analysis. a. DynaMine prediction of F57A flexibility. b. DynaMine prediction of flexibility of the CMT disease mutations in P2. Our residue numbering differs by − 1 from the mutation reports, to comply with the conventional numbering of residues in P2 and other FABP structures. c. Mapping of central residues onto the P2 structure. The structure shown is the liganded structure of the P38G mutant during MD simulations, to highlight the opening. The centralities were calculated from the unliganded crystal structures. The bound fatty acid is shown as a magenta surface and Phe57 in orange. Residues having high Z scores of centrality are indicated in blue, and the ones showing higher centrality in F57A than the wild-type P2 in yellow. Note how the opening β flap has no residues of high centrality (red circle). See Additional file 1: Figure S1 for more details