Structural studies of the C-terminal cysteine-rich domain of Aedes albopictus vitellogenin reveal an evolutionarily divergent, monomeric C-terminal cysteine knot (CTCK) architecture

Berlinguer, M.; Sadeghichehelgaz, M.; Vetrano, A.; D'Aquilio, A.; Mercuri, F.; Villa, M.; Gabrieli, P.; Iacobucci, C.; Forneris, F.

doi:10.64898/2026.05.25.727680

bioRxiv · 10.64898/2026.05.25.727680

Structural studies of the C-terminal cysteine-rich domain of Aedes albopictus vitellogenin reveal an evolutionarily divergent, monomeric C-terminal cysteine knot (CTCK) architecture

Berlinguer, M.·Sadeghichehelgaz, M.·Vetrano, A.·D'Aquilio, A.·Mercuri, F.·Villa, M.·Gabrieli, P.·Iacobucci, C.·Forneris, F.

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Abstract

Vitellogenins are essential transport lipoproteins and precursors to egg-yolk proteins in oviparous species. Several molecular structure studies have elucidated most of their multi-domain organization, yet the structure and function of their C-terminal cysteine knot (CTCK) domain have remained largely elusive. In this study, we present the 1.2 [A] resolution crystal structure of the recombinant CTCK domain from a vitellogenin isoform of the mosquito Aedes albopictus (Vg-CTCK). The molecular architecture reveals a CTCK fold defined by two antiparallel beta sheets stabilized by three intramolecular disulfide bonds, featuring a unique 12-amino acid insertion shaping an alpha helix positioned between the two main beta sheets. Analysis of the crystal packing and biophysical characterization in solution consistently confirms that recombinant Vg-CTCK is monomeric. To validate these findings in a native context, we employed ex vivo cross-linking mass spectrometry (XL-MS) on intact mosquito ovaries, which corroborated the molecular architecture of the Vg-CTCK observed in the crystal structure and highlighted the absence of inter-molecular cross-links. Collectively, our data highlight an evolutionarily divergent, monomeric assembly for the mosquito Vg-CTCK domain, challenging previous hypotheses that suggested this domain might facilitate vitellogenin oligomerization.

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Berlinguer, M., Sadeghichehelgaz, M., Vetrano, A., D'Aquilio, A., Mercuri, F., Villa, M., Gabrieli, P., Iacobucci, C., Forneris, F.. 2026-05-26. Structural studies of the C-terminal cysteine-rich domain of Aedes albopictus vitellogenin reveal an evolutionarily divergent, monomeric C-terminal cysteine knot (CTCK) architecture. https://doi.org/10.64898/2026.05.25.727680

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