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The cellular form of the prion protein (PrP C ) 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. PrP C 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 PrP C 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 PrP C (HuPrP) and NCAM interaction using stimulated emission depletion (STED) nanoscopy, SPR, and NMR spectroscopy approaches. PrP C co-localizes with NCAM in mouse hippocampal neurons, and this interaction is mainly mediated by the intrinsically disordered PrP C 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 PrP C N terminus as a dynamic and functional element responsible for protein-protein interaction.A misfolded form of the host-encoded cellular prion protein (PrP C ) 3 is the causative agent for a class of human and animal neurodegenerative diseases denoted as transmissible spongiform encephalopathies. PrP C is a sialoglycoprotein, tethered to the outer leaflet of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Soluble, natively ␣-helix-folded monomers of PrP C may adopt an aggregated protease-resistant conformation known as PrP Sc (1). The mature human PrP C (HuPrP) is composed of 209 residues including a largely unstructured N-terminal part and a globular ␣-helix-rich C-terminal domain (2). Conversely, PrP Sc is ␤-sheet-enriched, partially protease-resistant, insoluble, and multimeric (3). The insoluble nature of PrP Sc and its propensity to aggregate have hampered the use of high-resolution techniques, and therefore different PrP Sc models currently exist (4). Despite the fact that PrP C is highly conserved among different species, its physiological function has not been fully clarified. Defining PrP C function remains one of the main challenges in prion biology, and it is also an absolute requirement for understanding prion diseases. It is now being accepted that PrP C is a pivotal molecule with diverse roles in brain development and in neural plasticity in the adult (5-8). Proposed PrP C functions range from neuronal growth and differentiation (9), synaptic plasticity (10, 11), cell signaling (12, * This work was supported by the EC th...

The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.

Chronic wasting disease (CWD) is a highly infectious prion disease of cervids. Accumulation of prions, the disease-specific structural conformers of the cellular prion protein (PrPC), in the central nervous system, is the key pathological event of the disorder. The analysis of cervid PrPC sequences revealed the existence of polymorphism at position 226, in which deer PrP contains glutamine (Q), whereas elk PrP contains glutamate (E). The effects of this polymorphism on CWD are still unknown. We determined the high-resolution nuclear magnetic resonance structure of the mule deer prion protein that was compared to previously published PrP structures of elk and white-tailed deer. We found that the polymorphism Q226E could influence the long-range intramolecular interactions and packing of the β2−α2 loop and the C-terminus of the α3 helix of cervid PrP structures. This solvent-accessible epitope is believed to be involved in prion conversion. Additional differences were observed at the beginning of the well-defined C-terminus domain, in the α2−α3 region, and in its interactions with the α1 helix. Here, we highlight the importance of the PrP structure in prion susceptibility and how single amino acid differences might influence the overall protein folding.

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