Molecular mechanisms of Charcot-Marie-Tooth neuropathy linked to mutations in human myelin protein P2

Abstract

Charcot-Marie-Tooth (CMT) disease is one of the most common inherited neuropathies. Recently, three CMT1-associated point mutations (I43N, T51P, and I52T) were discovered in the abundant peripheral myelin protein P2. These mutations trigger abnormal myelin structure, leading to reduced nerve conduction velocity, muscle weakness, and distal limb atrophy. P2 is a myelin-specific protein expressed by Schwann cells that binds to fatty acids and membranes, contributing to peripheral myelin lipid homeostasis. We studied the molecular basis of the P2 patient mutations. None of the CMT1-associated mutations alter the overall folding of P2 in the crystal state. P2 disease variants show increased aggregation tendency and remarkably reduced stability, T51P being most severe. In addition, P2 disease mutations affect protein dynamics. Both fatty acid binding by P2 and the kinetics of its membrane interactions are affected by the mutations. Experiments and simulations suggest opening of the β barrel in T51P, possibly representing a general mechanism in fatty acid-binding proteins. Our findings demonstrate that altered biophysical properties and functional dynamics of P2 may cause myelin defects in CMT1 patients. At the molecular level, a few malformed hydrogen bonds lead to structural instability and misregulation of conformational changes related to ligand exchange and membrane binding.

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

“…For negatively stained EM, 740 µM DMPC:DMPG (1:1) SUVs were mixed with proteins using protein-to-lipid ratios of 1:58, 1:100, 1:200, and 1:500 and incubated at 22 °C for 1 h. EM grids were then prepared, stained and imaged as described before (Raasakka et al 2017; Raasakka et al 2019b; Ruskamo et al 2017).…”

Sub-atomic resolution crystal structures reveal conserved geometric outliers at functional sites

“…This local strain may be related to conformational changes taking place when P2 binds to a membrane surface and/or the portal region opens for fatty acid release. Such opening has thus far been observed in atomistic MD simulations and SAXS experiments [12,18].…”

“…The anisotropy of P2 has clear localization and direction, and these together hint at flexibility in the portal region corresponding to eventual open/close motions (Figure 1e). Opening of the portal region is a common feature proposed for the entire FABP family, and we recently observed such conformational changes in extended MD simulations [12,18]. The concentration of bent side-chain and main-chain conformations close to each other in space, especially around Arg78, is likely to be relevant for membrane or fatty acid binding-related conformational changes in P2.…”

“…P2 has a role in maintaining lipid homeostasis in the developing nervous system [7]. Mutations in the gene encoding P2 are linked to Charcot-Marie-Tooth disease [8][9][10][11][12]. We have previously determined the atomic-resolution 0.93-Å structure of human P2 [13], providing accurate insights into its ligand-binding determinants.…”

Determinants for forming a supramolecular myelin-like proteolipid lattice

“…Three CMT1-associated P2 protein variants have been characterized at the molecular level, showing altered fatty acid and lipid membrane binding properties. The most drastic CMT1 mutation, T51P, also reduced the membrane stacking capability of P2 30 . Overall, the stability of the mutant proteins was decreased, even though crystal structures indicated only minor structural changes compared to wild-type P2.…”