The Neurite Outgrowth Inhibitory Nogo-A-Δ20 Region Is an Intrinsically Disordered Segment Harbouring Three Stretches with Helical Propensity.
Functional recovery from central neurotrauma, such as spinal cord injury, is limited by myelin-associated inhibitory proteins. The most prominent example, Nogo-A, imposes an inhibitory cue for nerve fibre growth via two independent domains: Nogo-A-Δ20 (residues 544-725 of the rat Nogo-A sequence) and Nogo-66 (residues 1026-1091). Inhibitory signalling from these domains causes a collapse of the neuronal growth cone via individual receptor complexes, centred around sphingosine 1-phosphate receptor 2 (S1PR2) for Nogo-A-Δ20 and Nogo receptor 1 (NgR1) for Nogo-66. Whereas the helical conformation of Nogo-66 has been studied extensively, only little structural information is available for the Nogo-A-Δ20 region. We used nuclear magnetic resonance (NMR) spectroscopy to assess potential residual structural propensities of the intrinsically disordered Nogo-A-Δ20. Using triple resonance experiments, we were able to assign 94% of the non-proline backbone residues. While secondary structure analysis and relaxation measurements highlighted the intrinsically disordered character of Nogo-A-Δ20, three stretches comprising residues 561EAIQESL567, 639EAMNVALKALGT650, and 693SNYSEIAK700 form transient α-helical structures. Interestingly, 561EAIQESL567 is situated directly adjacent to one of the most conserved regions of Nogo-A-Δ20 that contains a binding motif for β1-integrin. Likewise, 639EAMNVALKALGT650 partially overlaps with the epitope recognized by 11C7, a Nogo-A-neutralizing antibody that promotes functional recovery from spinal cord injury. Diffusion measurements by pulse-field gradient NMR spectroscopy suggest concentration- and oxidation state-dependent dimerisation of Nogo-A-Δ20. Surprisingly, NMR and isothermal titration calorimetry (ITC) data could not validate previously shown binding of extracellular loops of S1PR2 to Nogo-A-Δ20.
Department of Physical Chemistry, ETH Zurich, Zurich, Switzerland.
Viviane
V
Zelenay
Brain Research Institute, University of Zurich, Zurich, Switzerland.|Department of Biology, ETH Zurich, Zurich, Switzerland.
Michael E
ME
Arzt
Department of Physical Chemistry, ETH Zurich, Zurich, Switzerland.
Stefan
S
Bibow
Brain Research Institute, University of Zurich, Zurich, Switzerland.|Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland.
Martin E
ME
Schwab
Department of Physical Chemistry, ETH Zurich, Zurich, Switzerland.
Roland
R
Riek
D000818:Animals; D002151:Calorimetry; D002460:Cell Line; D002942:Circular Dichroism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D009185:Myelin Proteins / Q000235:genetics / Q000378:metabolism; D009186:Myelin Sheath / Q000378:metabolism; D016501:Neurites / Q000378:metabolism / Q000502:physiology; D000071446:Neuronal Outgrowth / Q000235:genetics / Q000502:physiology*; D000070798:Nogo Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D051381:Rats; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D013119:Spinal Cord Injuries / Q000378:metabolism
D009185:Myelin Proteins; D000070798:Nogo Proteins