Solid-state NMR spectroscopy of membrane-associated myelin basic protein--conformation and dynamics of an immunodominant epitope

Abstract

Myelin basic protein (MBP) maintains the tight multilamellar compaction of the myelin sheath in the central nervous system through peripheral binding of adjacent lipid bilayers of oligodendrocytes. Myelin instability in multiple sclerosis (MS) is associated with the loss of positive charge in MBP as a result of posttranslational enzymatic deimination. A highly-conserved central membrane-binding fragment (murine N81-PVVHFFKNIVTPRTPPP-S99, identical to human N83-S101) represents a primary immunodominant epitope in MS. Previous low-resolution electron paramagnetic resonance measurements on the V83-T92 fragment, with Cys-mutations and spin-labeling that scanned the epitope, were consistent with it being a membrane-associated amphipathic alpha-helix. Pseudodeimination at several sites throughout the protein, all distal to the central segment, disrupted the alpha-helix at its amino-terminus and exposed it to proteases, representing a potential mechanism in the autoimmune pathogenesis of MS. Here, we have used magic-angle spinning solid-state NMR spectroscopy to characterize more precisely the molecular conformation and dynamics of this central immunodominant epitope of MBP in a lipid milieu, without Cys-substitution. Our solid-state NMR measurements have revealed that the alpha-helix present within the immunodominant epitope is shorter than originally modeled, and is independent of the pseudodeimination, highlighting the importance of the local hydrophobic effects in helix formation and stability. The main effect of pseudodeimination is to cause the cytoplasmic exposure of the fragment, potentially making it more accessible to proteolysis. These results are the first, to our knowledge, to provide atomic-level detail of a membrane-anchoring segment of MBP, and direct evidence of decreased MBP-membrane interaction after posttranslational modification.

Figure 1

(A) Structure of the myelin sheath. (B) The myelin sheath consists of stacked lipid bilayers. Myelin basic protein peripherally binds the adjacent leaflets on the cytoplasmic side and acts as an adhesive protein. (C) There are three potential amphipathic helices in MBP shown in the figure, which may peripherally interact with lipids and represent putative lipid binding domains. The details of protein-lipid binding are not known, and the arrangement shown in this figure is one out of many possibilities.

Figure 2

Amino-acid sequences of recombinant murine 18.5 kDa rmC1 and rmC8 MBP variants. (Dashed rectangles) Gln substitutions (pseudodeimination) performed to produce rmC8. The highlighted region is the fragment examined here using MAS-SSNMR.

Figure 3

The one-dimensional ¹³C spectra for rmC1 and rmC8 reconstituted in membranes of different lipid compositions and at different P/Ls. (A) The CPMAS DQF spectra for three rmC1 samples A–C (Table 1). (B) The CPMAS DQF spectra for two rmC8 samples D and E (Table 1). All spectra were acquired at −25°C and processed with a 100-Hz exponential window function before Fourier transformation. The spectra are scaled as indicated by the multiplication factors in both panels to bring them to equal heights for better visual comparison.

Figure 4

(A) One-dimensional ¹³C (CON)CA and (CANCO)CA spectra used to assign the C^α peaks of both Val⁸³ and Val⁸⁴ in membrane-associated rmC1. Both spectra were acquired at −25°C and processed with 100-Hz exponential line broadening before Fourier transformation. Because there is only one isotopically labeled pair of adjacent residues in the rmC1 and rmC8 sequences, both spectra should ideally display only one peak each. Because of scrambling during selective-labeling, there are many other overlapping residues that show up in the range from 52 to 62 ppm. (B) Two-dimensional (CO)NCOCA correlation spectrum allowing identification of the Asn⁸¹C^α chemical shift. (C) Two-dimensional 600 MHz ¹³C-¹³C correlation spectrum of membrane-associated rmC1 recorded at −25°C. (D) Sequence-corrected chemical shift deviations of C^α atoms of membrane-associated rmC1 and rmC8, at −25°C (shown in black). (Dark shaded bars) One standard deviation of α-helical ΔδC^α shifts. (Light shaded bars) One standard deviation of β-sheet ΔδC^α shifts.

Figure 5

(A) The Val C^α/C^β correlation region of a ¹³C-¹³C correlation spectrum as a function of temperature. V91 and V83 positions and V84 correlations are shown in the low temperature spectrum. (B) Projections of carbon correlations in the Val C^α/C^β region for both rmC1 and rmC8 at all four temperatures, showing redistribution of intensities from the Val⁸³/Val⁸⁴ C^α to the Val⁹¹ C^α region.

Figure 6

Two-dimensional ¹H-¹³C INEPT heteronuclear chemical-shift correlation spectra in [¹³-C-Asn,Val,U-¹⁵N]-labeled rmC1 and rmC8. (Red spectra) Samples with unlabeled protein, showing lipid correlations. The one-dimensional ¹H traces at the position of V91C^α are shown at the bottom for both spectra.

Figure 7

The immunodominant epitope adopts a similar α-helical molecular conformation in both rmC1 and rmC8 proteins. The α-helix is more exposed in rmC8, as follows from the dynamic information derived from the measurements of proton line widths.