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

Abstract

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.

Nap1 and Kap114 co-chaperone H2A-H2B and facilitate targeted histone release in the nucleus

“…Biochemical and structural studies of binary Kap114-H2A-H2B and Nap1 2 -H2A-H2B interactions, and pull-down studies of Kap114 and Nap1 from yeast lysates have been reported (13,22,36,41). First, we confirmed the previous findings using analytical ultracentrifugation (AUC), size exclusion chromatography multi-angle light scattering (SEC-MALS) and pull-down binding assays with recombinant Kap114, Nap1 and H2A-H2B (SI Appendix, Fig.…”

“…The superhelical Kap114 wraps around the H2A-H2B histone-fold domain (Fig. 1B), occluding the histone's DNA-binding surfaces and thus also acting as a histone chaperone (40,41). The Kap114•H2A-H2B complex is unusual in its interactions with the GTPase Ran GTP .…”

“…The Kap114•H2A-H2B complex is unusual in its interactions with the GTPase Ran GTP . Most importin•cargo complexes are dissociated by Ran GTP but Kap114•H2A-H2B binds Ran GTP to form a stable Ran GTP •Kap114•H2A-H2B complex, which alters interactions between Kap114 and H2A-H2B, and facilitates histone transfer to other nuclear chaperones or to the assembling nucleosome (41).…”

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

“…25 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. 53,54 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.…”

“…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, 53,54 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).…”