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

Abstract

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 beta-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 N-terminus 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 1H 15N NMR HSQC, hydrogen exchange, and 15N 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.

Protein folding at the membrane interface, the structure of Nogo-66 requires interactions with a phosphocholine surface

“…Although this domain is active, we find it to be highly flexible in an aqueous environment. Notably, other studies have found that most regions are disordered in the largest splicevariant, Nogo-A (1,192 amino acids) (14,15). Disordered structure is an interesting parallel feature between Nogo and another This article is a PNAS Direct Submission.…”

Resilience of death: intrinsic disorder in proteins involved in the programmed cell death

“…48 NMR analysis revealed that an important regulator of proliferation and apoptosis, cAMPresponsive (CRE)-binding (CREB) protein (CBP), contains an intrinsically disordered ACTR-binding domain (residues 2059-2117) that completely folds upon binding. 49 Combined experimental and computational analysis revealed that N-and C-terminal regions of the human Nogo proteins 50 and C-terminal domain of a mitochondrial pro-apoptotic protein ARTS 51 are typical IDPRs, and that the prostate apoptosis response factor-4 (PAR-4), 52 prostate-associated gene 4 (PAGE4) protein, 53 and important oncoprotein c-Myc 54 are mostly disordered apoptosis-related proteins.…”

Functions of reticulons in plants: What we can learn from animals and yeasts

“…To our knowledge, there is only fragmented data on this aspect. For instance, the implication of the six cysteine residues in the folding of bacterial-overexpressed soluble large extramembrane domain located at the N terminus of human RTN4A has been studied using CD and NMR spectroscopies [8,21]. The results showed that two cysteine residues (Cys699 and Cys912) out of the six, are not involved in a structurally disulfide bound.…”

“…For instance, AtRTNLB19 contains a 3b-hydroxysteroid dehydrogenase isomerase domain that functions in vitro as a 3b-hydroxysteroid dehydrogenase/C-4 decarboxylase activity when the RHD has been removed [7]. Beside the structured sequences, the C- and/or N-terminal domains can be highly unstructured, possibly due to the presence of proline and alanine residues [8]. This property seems important to form multiprotein complexes [9], and possibly to carry out alternative functions [10] as they can fold upon binding to their partners [11].…”