Identification and structural mechanism for a novel interaction between a ubiquitin ligase WWP1 and Nogo-A, a key inhibitor for central nervous system regeneration.
Nogo-A has been extensively demonstrated to play key roles in inhibiting central nervous system regeneration, regulating endoplasmic reticulum formation, and maintaining the integrity of the neuromuscular junction. In this study, an E3 ubiquitin ligase WWP1 was first identified to be a novel interacting partner for Nogo-A both in vitro and in vivo. By using CD, ITC, and NMR, we have further conducted extensive studies on all four WWP1 WW domains and their interactions with a Nogo-A peptide carrying the only PPxY motif. The results lead to several striking findings. (1) Despite containing an unstructured region, the 186-residue WWP1 fragment containing all four WW domains is able to interact with the Nogo-A(650-666) peptide with a high affinity, with a dissociation constant (K(d)) of 1.68 microM. (2) Interestingly, four isolated WW domains show differential structural properties in the free states. WW1 and WW2 are only partially folded, while WW4 is well-folded. Nevertheless, they all become well-folded upon binding to Nogo-A(650-666), with K(d) values ranging from 1.03 to 3.85 microM. (3) The solution structure of the best-folded WW4 domain is determined, and the binding-perturbed residues were derived for both WW4 and Nogo-A(650-666) by NMR HSQC titrations. Moreover, on the basis of the NMR data, the complex model is constructed by HADDOCK 2.0. This study provides rationales as well as a template Nogo-A(650-666) for further design of molecules to intervene in the WWP1-Nogo-A interaction which may regulate the Nogo-A protein level by controlling its ubiquitination.
Qin
Haina
H
Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.
Pu
Helen X
HX
Li
Minfen
M
Ahmed
Sohail
S
Song
Jianxing
J
D000595:Amino Acid Sequence; D000818:Animals; D002490:Central Nervous System / Q000378:metabolism*; D002942:Circular Dichroism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000737:chemistry* / Q000378:metabolism; D000070798:Nogo Proteins; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism
D009185:Myelin Proteins; D000070798:Nogo Proteins; C000603199:RTN4 protein, human; D025801:Ubiquitin; C471642:WWP1 protein, human; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase