Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

Abstract

Kinetochores assemble onto specialized centromeric CENP-A (centromere protein A) nucleosomes (CENP-A^Nuc) to mediate attachments between chromosomes and the mitotic spindle. We describe cryo-electron microscopy structures of the human inner kinetochore constitutive centromere associated network (CCAN) complex bound to CENP-A^Nuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-A^Nuc, and a linker DNA segment of the α-satellite repeat emerges from the fully wrapped end of the nucleosome to thread through the central CENP-LN channel that tightly grips the DNA. The CENP-TWSX histone-fold module further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the linker DNA by CCAN provides a robust mechanism by which kinetochores withstand both pushing and pulling forces exerted by the mitotic spindle.

Figure 1. CCAN is assembled from a network of interdependent modules.

(A, B) Cryo-EM density map (A) and molecular architecture (B) of the apo-CCAN^ΔCT fitted into the cryo-EM map with resolved OPQUR^Foot and HIK^Head. (C) Atomic model of the apo-CCAN^ΔCT with details of the CENP-N-CENP-M interface as well as CENP-O binding to the CENP-HI pocket shown as insets. (D) Cartoon schematic of the CCAN^ΔCT modules.

Figure 2. Structure of the CCAN^ΔT-CENP-A^Nuc complex.

(A) Right panel shows the consensus CCAN^ΔT-5 CENP-A^Nuc cryo-EM map (transparent white) overlaid onto the composite CCAN^ΔT-CENP-A^Nuc cryo-EM density map based on individual cryo-EM maps for the CENP-A^Nuc-CENP-C^N and CCAN^ΔT-DNA reconstructions. Left panel: composite map alone. (B) Two orthogonal views of the CCAN^ΔT-CENP-A^Nuc complex depicted in cartoon representation for protein and space filling for DNA. CENP-A^Nuc has a diameter of 11 nm.

Figure 3. The extranucleosomal linker DNA is gripped by the CENP-LN channel.

(A) Cartoon representation of CCAN^ΔT-CENP-A^Nuc with cryo-EM density shown for extranucleosomal linker DNA (from CCAN^ΔT-DNA cryo-EM density map). (B) The CENP-LN module features a positively-charged channel that complements the DNA duplex. Electrostatic surface charge shown for CENP-LN. (C and D). Details of the CENP-LN channel-DNA interaction. R169 of CENP-N inserts into the DNA minor groove. CENP-LN surface charge shown in C. (E) Cartoon schematic of the CCAN^ΔT modules showing DNA path. (F and G) CENP-C binds to two sites on CCAN. The DEFxIDE motif binds to CENP-LN (E), whereas the FxxLFL motif binds to CENP-HIKM (G).

Figure 4. Architecture of the full CCAN^ΔC-DNA complex.

(A) Cryo-EM density map, and (B) molecular model of the CCAN^ΔC-DNA complex with DNA map density shown in yellow. (C) CCAN assembles an enclosed chamber that topologically entraps linker DNA. (D) Electrostatic representation of the DNA-binding chamber formed by CENP-LN, CENP-TW and CENP-HIK^Head modules shows a positively charged chamber that tightly grips DNA. (E) Schematic of CCAN showing DNA path through the CENP-LN, CENP-HIKM and CENP-TWSX modules. (F) The CENP-TWSX module resembles the canonical H3/H4 nucleosome tetramer, and partially wraps DNA.

Figure 5. Structure of the complete CCAN-CENP-A inner kinetochore module.

(A) Atomic model of the CCAN-CENP-A^Nuc complex. (B) Cartoon schematic of the CCAN-CENP-A^Nuc complex. (C) Mutations of CENP-N and CENP-TW previously shown to impair centromere function contribute to the DNA-binding channel of the CCAN (, –9). Residues implicated in DNA binding by CENP-L, and mutated in the current study, are also shown.