Fahoum, J. | bioRxiv Science

bioRxiv2024

Binding of SARS-CoV-2 nucleocapsid protein to uninfected epithelial cells induces antibody-mediated complement deposition

SARS-CoV-2 infection triggers strong antibody response toward Nucleocapsid-Protein (NP), suggesting extracellular presence beyond its intra-virion RNA binding. Interestingly, NP was found to decorate infected and proximal uninfected cell-surfaces. Here, we propose a new mechanism through which extracellular NP on uninfected cells contributes to COVID-19 pathogenicity. We show that NP binds to cell-surface sulfated linear-glycosaminoglycans by spatial rearrangement of its RNA-binding sites facilitated by the flexible, positively charged, linker. Coating of uninfected lung-derived cells with purified NP attracted anti-NP-IgG from lung fluids and sera collected from COVID-19 patients. The magnitude of this immune recognition was significantly elevated in moderate compared to mild COVID-19 cases. Importantly, binding of anti-NP-IgG present in sera generated clusters that triggered C3b deposition by the classical complement pathway. Heparin analog enoxaparin outcompeted NP-binding, rescuing cells from anti-NP IgG-mediated complement deposition. Our findings unveil how extracellular NP may exacerbate COVID-19 tissue damage, and suggest leads for preventative therapy. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/585388v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1084e20org.highwire.dtl.DTLVardef@4126caorg.highwire.dtl.DTLVardef@159c610org.highwire.dtl.DTLVardef@da7b7f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIIgG from patients sera target NP-bound cells resulting in complement activation C_LIO_LIThe flexible linker allows NP to both bind linear sulfated GAGs and wrap around RNA C_LIO_LIHeparin analogs prevent NP surface binding and alleviate complement activation C_LIO_LICell-ELISA anti-NP IgG levels differ between mild and moderate COVID-19 C_LI

immunology↗

bioRxiv2021

Tandem WW/PPxY motif interactions in WWOX: the multifaceted role of the second WW domain

Class I WW domains mediate protein interactions by binding short linear PPxY motifs. They occur predominantly as tandem repeats, and their target proteins often contain multiple PPxY motifs, but the interplay of WW/peptide interactions is not always intuitive. WW domain-containing oxidoreductase (WWOX) protein harbors two WW domains: unstable WW1 capable of PPxY binding, and well-folded but mutated WW2 that cannot bind such motifs. WW2 is considered to act as a WW1 chaperone, but the underlying mechanism remains to be revealed. Here we combine NMR, ITC and structural modeling to elucidate the role of both WW domains in WWOX binding to single and double motif peptides derived from its substrate ErbB4. Using NMR we identified an interaction surface between the two domains that supports a WWOX conformation that is compatible with peptide substrate binding. ITC and NMR measurements reveal that while binding affinity to a single motif is marginally increased in the presence of WW2, affinity to a dual motif peptide increases tenfold, and that WW2 can directly bind double motif-peptides using its canonical binding site. Finally, differential binding of peptides in a mutagenesis study is consistent with a parallel orientation binding to the WW1-WW2 tandem domain, agreeing with structural models of the interaction. Our results reveal the complex nature of tandem WW domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both stability and binding. This opens the way to assess how evolution can utilize the multivariate nature of binding to fine-tune interactions for specific biological functions.

biophysics↗