title: Mutation of Ser-50 and Cys-66 in Snapin modulates protein structure and stability.
abstract: Snapin is a 15 kDa protein present in neuronal and non-neuronal cells that has been implicated in the regulation of exocytosis and endocytosis. Protein kinase A (PKA) phosphorylates Snapin at Ser-50, modulating its function. Likewise, mutation of Cys-66, which mediates protein dimerization, impairs its cellular activity. Here, we have investigated the impact of mutating these two positions on protein oligomerization, structure, and thermal stability, along with the interaction with SNARE proteins. We found that recombinant purified Snapin in solution appears mainly as dimers in equilibrium with tetramers. The protein exhibits modest secondary structure elements and notable thermal stability. Mutation of Cys-66 to Ser abolished subunit dimerization, but not higher-order oligomers. This mutant augmented the presence of α-helical structure and slightly increased the protein thermal stability. Similarly, the S50A mutant, mimicking the unphosphorylated protein, also exhibited a higher helical secondary structure content than the wild type, along with greater thermal stability. In contrast, replacement of Ser-50 with Asp (S50D), emulating the protein-phosphorylated state, produced a loss of α-helical structure, concomitant with a decrease in protein thermal stability. In vitro, the wild type and mutants weakly interacted with SNAP-25 and the reconstituted SNARE complex, although S50D exhibited the strongest binding to the SNARE complex, consistent with the observed higher cellular activity of PKA-phosphorylated Snapin. Our observations suggest that the stronger binding of S50D to SNAREs might be due to a destabilization of tetrameric assemblies of Snapin that favor the interaction of protein dimers with the SNARE proteins. Therefore, phosphorylation of Ser-50 has an important impact on the protein structure and stability that appears to underlie its functional modulation.
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche, Spain.
authors.forename: Aaron
authors.initials: A
authors.lastname: Navarro
authors.forename: José A
authors.initials: JA
authors.lastname: Encinar
authors.forename: Blanca
authors.initials: B
authors.lastname: López-Méndez
authors.forename: David
authors.initials: D
authors.lastname: Aguado-Llera
authors.forename: Jesús
authors.initials: J
authors.lastname: Prieto
authors.forename: Javier
authors.initials: J
authors.lastname: Gómez
authors.forename: Luís Alfonso
authors.initials: LA
authors.lastname: Martínez-Cruz
authors.forename: Oscar
authors.initials: O
authors.lastname: Millet
authors.forename: José Manuel
authors.initials: JM
authors.lastname: González-Ros
authors.forename: Gregorio
authors.initials: G
authors.lastname: Fernández-Ballester
authors.forename: José L
authors.initials: JL
authors.lastname: Neira
authors.forename: Antonio
authors.initials: A
authors.lastname: Ferrer-Montiel
mesh_terms: D003545:Cysteine / Q000235:genetics*; D009154:Mutation; D011487:Protein Conformation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D050600:SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics*; D017550:Spectroscopy, Fourier Transform Infrared; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D012694:Serine; D003545:Cysteine