Molecular basis of RanGTP-activated nucleosome assembly with Histones H2A-H2B bound to Importin-9

Abstract

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.

Mechanism of RanGTP priming H2A-H2B release from Kap114 in an atypical RanGTP•Kap114•H2A-H2B complex

“…H2A-H2B release becomes targeted as conformational changes in RanGTP•Kap114•H2A-H2B release contacts between H2A-H2B and the N-terminal HEAT repeats of Kap114 to make H2A-H2B available for interactions with nucleosomal DNA and H3-H4. These findings are validated and extended to Imp9 by solution HDX analysis ( 30 ).…”

“…We have revealed how Kap114 chaperones H2A-H2B in the cytoplasm and in the nucleus, the latter in the form of the RanGTP•Kap114•H2A-H2B complex. Our previous study of Imp9 binding to H2A-H2B and solution HDX analysis of Imp9 show that this importin uses the same mechanism ( 30 ). This knowledge is the foundation to understand how additional molecular players contribute to H2A-H2B cytoplasmic processing and deposition into the nucleosome.…”

Cryo-EM of the Nucleosome Core Particle Bound to Ran-RCC1 Reveals a Dynamic Complex

“…The complexation leads to a conformational change in importin 9/Kap114 that promotes the release of H2A-H2B into the assembling nucleosome. As importin-9 binds to H2A-H2B via a mechanism also involving arginine interactions with the acidic patch as well as to Ran, , it is conceivable that, in the vicinity of chromatin, a high concentration of RCC1 may result in competition between importins and RCC1 for binding to both H2A-H2B and Ran. The Ran-nucleosome interactions mentioned above are not observed in our structure, which features Ran in its (mostly) nucleotide-free state (see below).…”

“…In intact nuclei from human osteosarcoma U2OS cells, Ran was cross-linked to histones H2B, H3, and H4 . Additionally, Ran•GTP was found to form a complex with H2A-H2B and the histone chaperone importin 9 (as well as its yeast homologue Kap114) during nucleosome assembly. , In these ternary complexes, Ran•GTP modulates importin-histone interactions and makes transient contacts with α3 and αC of H2A. The complexation leads to a conformational change in importin 9/Kap114 that promotes the release of H2A-H2B into the assembling nucleosome.…”