Intrinsic Disorder of the Neuronal SNARE Protein SNAP25a in its Pre-fusion Conformation

• Published in: Journal of molecular biology 2023-05, Vol.435 (10), p.168069-168069, Article 168069
• Main Authors: Stief, Tobias, Gremer, Lothar, Pribicevic, Sonja, Espinueva, Delane F., Vormann, Katharina, Biehl, Ralf, Jahn, Reinhard, Pérez-Lara, Ángel, Lakomek, Nils-Alexander
• Format: Article
• Language: English
• Subjects: exocytosis; Humans; intrinsically disordered proteins; Membrane Fusion; molecular biology; neurons; Neurons - metabolism; NMR spectroscopy; nuclear magnetic resonance spectroscopy; Protein Conformation; protein dynamics; Scattering, Small Angle; small-angle X-ray scattering; SNAP25a; SNARE proteins; SNARE Proteins - metabolism; synaptic vesicles; X-Ray Diffraction
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2023.168069

[Display omitted] •Residue-specific structural insights on the SNARE protein SNAP25a by NMR.•The monomeric pre-fusion form of SNAP25 is primarily intrinsically disordered.•SNAP25a comprises an N-terminal α-helical region overlapping with the first SNARE motif.•The N-terminal helical region may act as a nucleation point for SNARE complex assembly. The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments. SAXS data indicate that monomeric SNAP25 is more compact than a Gaussian chain but still a random coil. NMR shows that for monomeric SNAP25a, before SNAP25a interacts with its SNARE partners to drive membrane fusion, only the N-terminal part (region A5 to V36) of the first SNARE motif, SN1 (L11 - L81), is helical, comprising two α-helices (ranging from A5 to Q20 and S25 toV36). From E37 onwards, SNAP25a is mostly disordered and displays high internal flexibility, including the C-terminal part of SN1, almost the entire second SNARE motif (SN2, N144-A199), and the connecting loop region. Apart from the N-terminal helices, only the C-termini of both SN1 (E73 - K79) and SN2 (region T190 - A199), as well as two short regions in the connecting loop (D99 - K102 and E123 - M127) show a weak α-helical propensity (α-helical population