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. 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 the ternary RanGTP•Importin-9•H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for release. 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, to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9•RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9•H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP•Kap114•H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome or to dedicated H2A-H2B chaperones in the nucleus.

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

“…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.…”

“…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).…”

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).…”