biostudies-literatureHernandez GSR&TDPPBIEuropean EC Horizon 2020MOSTMICRO FCTCOMPETE 2020FCTFoundation for Science and TechnologyFEDERSR&TDPT-NMR FCTLS4FUTUREgkaf502https://www.ebi.ac.uk/biostudies/studies/S-EPMC12159747biostudies-literatureUnknown53(11)The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral life cycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo phase separation is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein's dynamic self-assembly. We show that the N-protein's interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that thNucleic acids researchDynamic ensembles of SARS-CoV-2 N-protein reveal head-to-head coiled-coil-driven oligomerization and phase separation.PMC12159747CEECIND/01443/2017UIDP/04612/2020PD/BD/147227/2019UIDB/04612/2020810856PTDC/BIA-BFS/0391/20210145-FEDER-007660LA/P/0087/2020UI/BD/154576/202201/SAICT/2016Hernandez GGomes TVeloso TCordeiro TNLopes JFernandes NPMartins MLfalseDynamic ensembles of SARS-CoV-2 N-protein reveal head-to-head coiled-coil-driven oligomerization and phase separation.The SARS-CoV-2 nucleocapsid (N) protein is essential for the viral life cycle, facilitating RNA packaging, replication, and host-cell interactions. Its ability to self-assemble and undergo phase separation is critical for these functions but remains poorly understood. Using an integrated approach combining small-angle X-ray scattering (SAXS), nuclear magnetic resonance spectroscopy, computational modeling, and biophysical assays, we uncover key mechanisms underpinning N-protein's dynamic self-assembly. We show that the N-protein's interdomain linker (IDL) contains a conserved coiled-coil (CC) motif that drives transient interactions between protein subunits, enabling the formation of progressively larger complexes at higher concentrations. SAXS analysis and ensemble modeling reveal that th2025-01-01T00:00:00Z2025 Jun2026-06-03T07:31:01.5Z2026-04-26T03:09:26.818ZS-EPMC121597474050368610.1093/nar/gkaf502