The N- and C-termini of the human Nogo molecules are intrinsically unstructured: Bioinformatics, CD, NMR characterization, and functional implications

RTN4 or Nogo, proteins are composed of three alternative splice forms, namely 1192-residue Nogo-A, 373-residue Nogo-B, and 199-residue Nogo-C. Nogo, proteins have received intense attentions because they have been implicated in a variety of critical cellular processes including CNS neuronal regeneration, vascular remodeling, apoptosis, interaction with P-amyloid protein converting enzyme, and generation/maintenance of the tubular network of the endoplasmic reticulum (ER). Despite their significantly-different N-terminal lengths, they share a conserved C-terminal reticulon-homology domain consisting of two transmembrane fragments, a 66-residue extracellular loop Nogo-66 and a 38-residue C-tail carrying ER retention motif. Nogo-A owns the largest N-terminus with 1016 residues while the Nogo-B has an Nterminus almost identical to the first 200 residues of Nogo-A. So far, except for our previous determination of the Nogo-66 solution structure, no structural characterization of the other Nogo regions has been reported. In the present study, we initiated a systematically investigation of structural properties of Nogo, molecules by a combined use of bioinformatics, CD, and NMR spectroscopy. The results led to two striking findings: (1) in agreement with bioinformatics prediction, the N- and C-termini of Nogo-B were experimentally demonstrated to be intrinsically unstructured by CD, two-dimensional H-1 N-15 NMR HSQC, hydrogen exchange, and N-15 heteronuclear NOE characterization. (2) Further studies showed that the 1016-residue N-terminus of Nogo-A was again highly disordered. Therefore, it appears that being intrinsically-unstructured allows Nogo, molecules to serve as double-faceted functional players, with one set of functions involved in cellular signaling processes essential for CNS neuronal regeneration, vascular remodeling, apoptosis and so forth and with another in generating/maintaining membrane-related structures. We propose that this mechanism may represent a general strategy to place the formation/ maintenance of membrane-related structures under the direct regulation of the cellular signaling.