Human myelin protein P2: From crystallography to time-lapse membrane imaging and neuropathy-associated variants

Abstract

AbstractPeripheral myelin protein 2 (P2) is a fatty acid-binding protein expressed in vertebrate peripheral nervous system myelin, as well as in human astrocytes. Suggested functions of P2 include membrane stacking and lipid transport. Mutations in the PMP2 gene, encoding P2, are associated with Charcot-Marie-Tooth disease (CMT). Recent studies have revealed three novel PMP2 mutations in CMT patient families. To shed light on the structure and function of the corresponding P2 variants, we used X-ray and neutron crystallography, small-angle X-ray scattering, circular dichroism spectroscopy, computer simulations, and lipid binding assays. The crystal and solution structures of the I50del, M114T, and V115A variants of P2 showed only minor differences to the wild-type protein, whereas the thermal stability of the disease variants was reduced. Lipid vesicle aggregation assays revealed no change in membrane stacking characteristics, while the variants showed slightly altered fatty acid binding. Time-lapse imaging of lipid bilayers indicated membrane blebbing induced by P2, which could be related to its function in stacking of two curved membrane surfaces in myelin in vivo. All variants caused blebbing of membranes on similar timescales. In order to better understand the links between structure, dynamics, and function, the crystal structure of perdeuterated P2 was refined from room temperature data collected using both neutrons and X-rays, and the results were compared to molecular dynamics simulations and cryocooled crystal structures. Taken together, our data indicate similar properties of all known CMT variants of human P2; while crystal structures are nearly identical, stability and function of the disease variants are impaired compared to the wild-type protein. Our data provide new insights into the structure-function relationships and dynamics of P2 in health and disease.