To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of β-coronaviruses: SARS-CoV-2 S2 apex residues 980–1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

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Previously we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus (Padavannil et al. 2019). However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9•H2A-H2B complex and formation of RanGTP•Importin-9•H2A-H2B facilitates H2A-H2B release to the assembling nucleosome (Padavannil et al. 2019). Here we show cryo-EM structures of Importin-9•RanGTP and of its yeast homolog Kap114, including Kap114•RanGTP, Kap114•H2A-H2B, and RanGTP•Kap114•H2A-H2B. In combination with hydrogen-deuterium exchange analysis of Importin-9 complexes and nucleosome assembly assays, we explain how the conserved Kap114/Importin-9 importins bind H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. We show that RanGTP binds to the N-terminal repeats of Kap114/Importin-9 as in Kap114/Importin-9•RanGTP, and H2A-H2B binds via its acidic patch to the Kap114/Importin-9 C-terminal repeats as in Kap114/Importin-9•H2A-H2B. RanGTP-binding in RanGTP•Kap114•H2A-H2B changes Kap114/Importin-9 conformation such that it no longer contacts the surface of H2A-H2B proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome. The reduced affinity of RanGTP for Kap114/Importin-9 when H2A-H2B is bound may ensure release of H2A-H2B only at chromatin.

Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9·H2A-H2B complex, this RanGTP·Imp9·H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP·Imp9·H2A-H2B ternary complex to those in the Imp9·H2A-H2B or Imp9·RanGTP binary complexes. Our data are consistent with the Imp9·H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin·RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9.

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