Fuzzy complexes of myelin basic protein: NMR spectroscopic investigations of a polymorphic organizational linker of the central nervous system

Abstract

The classic 18.5 kDa isoform of myelin basic protein (MBP) is central to maintaining the structural homeostasis of the myelin sheath of the central nervous system. It is an intrinsically disordered, promiscuous, multifunctional, peripheral membrane protein, whose conformation adapts to its particular environment. Its study requires the selective and complementary application of diverse approaches, of which solution and solid-state NMR spectroscopy are the most powerful to elucidate site-specific features. We review here several recent solution and solid-state NMR spectroscopic studies of 18.5 kDa MBP, and the induced partial disorder-to-order transitions that it has been demonstrated to undergo when complexed with calmodulin, actin, and phospholipid membranes.

Fig. 1

Solution NMR spectroscopy of 18.5 kDa recombinant murine MBP (176 residues including an LEH₆ tag) in association with Ca²⁺-CaM (Libich and Harauz 2008b). (A) The ¹H-¹⁵N HSQC spectrum of ¹⁵N-labelled MBP in the presence of Ca²⁺-CaM. Of the 164 expected backbone peaks, 145 were assigned. (B) Secondary structure propensity (SSP) of MBP alone (top panel) and bound with CaM (bottom panel). The SSP score is interpreted as the percentage of conformers in the disordered ensemble that exhibit ordered secondary structure at a given residue (Marsh et al. 2006). The redistribution of propensities of structural conformers in the C-terminus of MBP indicates increased stabilization resulting from the binding of Ca²⁺-CaM. (Adapted from Libich and Harauz (2008b) with permission.)

Fig. 2

Solid-state NMR spectroscopy of 18.5 kDa recombinant murine MBP in association with actin filaments and bundles (Ahmed et al. 2009). (A) The ¹³C CP-MAS spectra and ¹³C INEPT-MAS spectra of ¹³C,¹⁵N-labelled MBP in complex with actin shown as a function of temperature. (B) Aliphatic region of TOBSY (total through-bond correlation spectroscopy) ¹³C–¹³C correlation spectrum of MBP in complex with actin collected at 800 MHz. (C) Expansions of the Ala C^α–C^β region, the Thr C^β–C^γ region, and the Pro/Val C^α–C^β region. The dashed squares indicate random coil regions, and arrows indicate the trends of the change in chemical shift as a function of type of secondary structure (α helix or β strand). The chemical shifts observed in the TOBSY spectrum are compared with the predominantly random coil chemical shifts observed for isolated MBP dissolved in 100 mmol·L⁻¹ KCl (Libich et al. 2007). (Adapted from Ahmed et al. (2009) with permission.)

Fig. 3

Solid-state NMR spectroscopy of 18.5 kDa recombinant murine MBP in association with DMPC:DMPG membranes (Zhong et al. 2007). (A) Schematic representation of the triple-resonance experiments used to obtain sequential assignments of lipid bilayer-associated ¹³C,¹⁵N-MBP. Light-grey boxes enclose correlated residues with one-bond and two-bond (N[i]-C^α[i-1]) polarization transfers indicated by solid and dashed arrows, respectively. (B) Two-dimensional ¹³C-¹³C planes extracted from the three-dimensional NCOCX and NCACX experiments. Sequence-specific assignments are indicated by the solid arrows. (C) Chemical shift index analysis of assigned C^α resonances and ¹⁵N line-width analysis for assigned residues. Tentatively-assigned residues in both panels are shown in grey. (Adapted from Zhong et al. (2007) with permission.)

Fig. 4

Summary of solution and solid-state NMR spectroscopic investigations of 18.5 kDa MBP. (A) The highly charged (+19 at neutral pH) recombinant murine version of the unmodified C1 component (rmC1) has been used as the prototype for the family. The C-terminal LEH₆ tag is not shown here. (B) Solution NMR spectroscopic studies of MBP in 30% TFE-d₂ defined 3 specific regions with strong α-helical propensity: Thr33–Asp46, Val83–Thr92, and Tyr142–Leu154 (Libich and Harauz 2008b; Harauz and Libich 2009). (C) Solution NMR investigations demonstrated that the C-terminal segment was the primary binding site for calmodulin in solution, as indicated by contiguous chemical shift perturbations (Pro120–Arg160) (Libich and Harauz 2008b). (D) Solid-state NMR studies of MBP-actin complexes indicated some induced disorder-to-order transitions in both the N- and C-terminal regions, as well as in the central immunodominant epitope (Ahmed et al. 2009). (E and F) Solid-state NMR spectroscopic investigations of MBP reconstituted with artificial membranes (Zhong et al. 2007) showed that part of the protein was immobilized by the interaction with the membrane (E), but a large portion of it was mobile (F).