Abstract
The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals. A misfolded, pathogenic isoform, denoted as prion, is related to a class of neurodegenerative diseases known as transmissible spongiform encephalopathy. PrPC function has not been unequivocally clarified, and it is rather defined as a pleiotropic protein likely acting as a dynamic cell surface scaffolding protein for the assembly of different signaling modules. Among the variety of PrPC protein interactors, the neuronal cell adhesion molecule (NCAM) has been studied in vivo, but the structural basis of this functional interaction is still a matter of debate. Here we focused on the structural determinants responsible for human PrPC (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrPC co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrPC N-terminal tail, which binds with high affinity to the NCAM fibronectin type-3 domain. NMR structural investigations revealed surface-interacting epitopes governing the interaction between HuPrP N terminus and the second module of the NCAM fibronectin type-3 domain. Our data provided molecular details about the interaction between HuPrP and the NCAM fibronectin domain, and revealed a new role of PrPC N terminus as a dynamic and functional element responsible for protein-protein interaction.
Highlights
The cellular form of the prion protein (PrPC) is a highly conserved glycoprotein mostly expressed in the central and peripheral nervous systems by different cell types in mammals
neuronal cell adhesion molecule (NCAM) belongs to Ig superfamily cell adhesion molecules (CAMs), and it is present on the cell surface of neurons, astrocytes, and oligodendrocytes, where it mediates homophilic and heterophilic cell adhesion [24]
NCAM Co-localizes with PrPC—Functional interactions of PrPC with its binding partner(s) have been suggested previously [13]: cis and trans interactions between NCAM and PrPC promote neurite outgrowth, and the disruption of these interactions indicates that PrPC is involved in nervous system development cooperating with NCAM as a signaling receptor
Summary
PrP, prion protein; HuPrP, human prion protein; MoPrP, mouse prion protein; BCL, short peptide from neuronal cell adhesion molecule; FNIII1,2, two fibronectin type-3 domains; FNIII1, first module of fibronectin type-3 domain; FNIII2, second module of fibronectin type-3 domain; NCAM, neuronal cell adhesion molecule; GAG, glycosaminoglycan; GPI, glycosylphosphatidylinositol; GC, growth cone(s); STED, stimulated emission depletion nanoscopy; HSQC, heteronuclear single quantum correlation; TOCSY, total correlation spectroscopy; RMSD, root mean square deviation. Despite the inherent structural disorder of the PrPC N-terminal domain, which can mediate its interaction with both metals and cellular polyanions (e.g. sulfate proteoglycans) [31], binding with physiological protein partners has not been proved yet. We examined the effect of a pathological amino acid substitution, P102L, the prototypical mutation linked to a genetic form of human prion disease denoted as Gerstmann-Straussler-Scheinker disease (GSS), on binding to FNIII2. This mutation unveiled a new hidden binding site corresponding to the non-octarepeat copper-binding site [32]. The novel insight of the effect of HuPrP bearing the pathogenic point mutation P102L on its interaction with the FNIII2 domain is introduced here
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