Fig 1.
(a) The sequence of the wt-P0ct construct, corresponding to amino acids 180–248 of the human P0 precursor, with an extra N-terminal Gly residue (gray) left behind from affinity tag cleavage. The Cys182 palmitoylation site was mutated into a Leu (green) in all constructs. Putative serine phosphorylation sites are indicated with asterisks. Residues affected by disease mutations are in bold. CMT1B, CMT2I, and DSS point mutations are shown in blue, red, and orange, respectively. The sequence highlighted in yellow corresponds to the neuritogenic segment used in EAN models [7]. Secondary structure prediction is shown below. (b) SEC traces of wt-P0ct and mutants as determined using a Superdex 75 10/300GL column. Note the slightly lower retention volume of D224Y, for which the chromatography had to be performed with a different running buffer than for the other variants. The degradation products (red asterisk) present with D224Y could be completely removed using SEC. The final purity of each P0ct variant (4 μg per lane) as determined using SDS-PAGE is shown as inset. (c) DLS data of P0ct variants display good monodispersity with minimal variation in Rh.
Table 1.
Recombinant protein characterization.
Fig 2.
SAXS analysis of P0ct in solution.
(a) SAXS data for P0ct variants. The scattering curves have been offset for clarity. (b) Guinier fits based on SAXS data. Data range is shown within each graph. (c) Distance distributions. (d) Kratky plots. P0ct variant data point coloring is consistent throughout the figure. GNOM fits to the data are shown as black lines in panels (a) and (c).
Fig 3.
Folding and lipid binding analysis of P0ct variants.
The folding of wt-P0ct and mutants was studied using SRCD spectropolarimetry in (a) water, (b) 30% TFE, (c) 0.5% SDS, (d) DMPC:DMPG (1:1), and (e) DMPC:DMPG (4:1) at 1:200 P/L ratio in each lipid condition. Additional spectra are presented in S2 Fig. (f) SPR measurements were used to determine the affinity of each P0ct variant to immobilized DMPC:DMPG (1:1) vesicles. The colour coding legend in panel (a) for each mutant trace also corresponds to all other traces in subsequent panels.
Table 2.
SPR fitting parameters.
Fig 4.
Analysis of protein-induced lipid structure behaviour.
(a) DSC analysis of lipid phase transition. The experiments were carried out at 350 μM DMPC:DMPG (1:1) and a 1:100 P/L ratio. (b) Turbidimetric analysis of 0.5 mM DMPC:DMPG (1:1) at 5 μM (gray) and 10 μM protein concentration (dark red). These proteins concentrations translate to 1:100 and 1:50 P/L ratios, respectively. Error bars represent standard deviation. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test to wt-P0ct turbidity within the same protein concentration series (*: P < 0.05; ***: P < 0.001). (c) SAXD analysis reveals that D224Y displays a significantly tighter mean repeat distance compared to wt-P0ct, whereas K236E is most loose. The traces have identical colouring to (a).
Fig 5.
Negatively stained samples of DMPC:DMPG (1:1) vesicles were imaged (a) alone, and with (b) wt-P0ct, (c) T216ER, (d) A221T, (e) D224Y, (f) R227S, (g) K236E, and (h) K236del at a 1:200 P/L ratio. D224Y forms multilayered lipid structures that are absent for wt-P0ct.
Fig 6.
SRCD stopped-flow kinetics of protein-induced initial lipid turbidification.
(a) The SRCD signal at 195 nm was monitored using rapid kinetics for 5 s. wt-P0ct and mutants were mixed with DMPC:DMPG (1:1) lipids at 1:200 P/L ratio in the presence of 150 mM NaF. Fits (dashed lines) are plotted over the measurement points. Error bars represent standard deviation. See Table 4 for fitting results. (b) Graphical comparison of the obtained k1 values.
Table 3.
Kinetic constants for protein-induced vesicle turbidity.
The kinetic constants were obtained by fitting the data to a two-phase exponential decay function. All errors represent standard deviation.
Fig 7.
(a) NR data for a supported DMPC:DMPG (1:1) bilayer before (open markers) and after incubation with wt-P0ct (closed markers). The solvent contrasts used were 95% D2O (red), Si-matched water (SMW, 38% D2O; green), and 100% H2O (blue). The error bars denote standard deviation. Fits are shown as dashed and solid lines for the bilayer before and after addition of wt-P0ct, respectively. (b) Scattering length density (ρ) profiles obtained from the fitting. (c) Model for the P0ct-bound membrane. The protein-free membrane is shown in light gray on the background.
Table 4.
The fits and obtained scattering length density profiles are shown in Fig 7.
Fig 8.
(a) Alignment of P0ct from selected vertebrates. The locations of the P0ct mutations are indicated with arrowheads. (b) ANCHOR2/IUPred2 analysis of P0ct highlights a region likely to fold upon ligand binding between residues 215–230. (c) Stereo view of a model of the predicted membrane-binding helical region. Hydrogen bonds are shown with thin blue lines.
Fig 9.
(a) Tentative models of the membrane-embedded P0ct helix with the two mutations having opposite effects on membrane stacking. The tilted orientation is estimated based on earlier oriented CD experiments on wt-P0ct [5]. (b) MD simulations starting from a helical structure in water. Blue, α helix; orange, coil; green, turn; light blue, β strand; yellow, 310 helix. (c) A working model for the binding of P0ct to the membrane cytosolic surface, based on current knowledge and predictions. It is likely that the C-terminal region, including Lys236, is less tightly bound to the surface. Positions of the mutations studied in the current work are indicated with red arrows.