Conserved Tetramer Junction in the Kinetochore Ndc80 Complex

Abstract

The heterotetrameric Ndc80 complex establishes connectivity along the principal longitudinal axis of a kinetochore. Its two heterodimeric subcomplexes, each with a globular end and a coiled-coil shaft, connect end-to-end to create a ∼600 Å long rod spanning the gap from centromere-proximal structures to spindle microtubules. Neither subcomplex has a known function on its own, but the heterotetrameric organization and the characteristics of the junction are conserved from yeast to man. We have determined crystal structures of two shortened ("dwarf") Ndc80 complexes that contain the full tetramer junction and both globular ends. The junction connects two α-helical coiled coils through regions of four-chain and three-chain overlap. The complexity of its structure depends on interactions among conserved amino-acid residues, suggesting a binding site for additional cellular factor(s) not yet identified.

Keywords: X-ray crystallography; cell division; coiled-coil junction; kinetochore assembly; kinetochore structure.

Figure 1

Ndc80c. (A) Schematic of Ndc80c. Upper diagram: Ndc80c was shortened by joining the globular ends of each heterodimer (Ndc80:Nuf2 or Spc24:Spc25) to the beginning of a heptad in the CC region, as illustrated by the dashed line. Lower diagram: The shortened Ndc80c retains the junction (J). Color code, used in all figures: Ndc80, red, Nuf2, orange, Spc24, green, Spc25, blue. (B) Polypeptide chains of Ndc80c^dwarf (left) and Ndc80c^e-dwarf (right). Numbers indicate boundaries determined from the crystal structures. The two additional heptads in Scp24^e-dwarf and Spc25^e-dwarf are in blue and yellow, respectively. Polypeptide chains of Ndc80c^e-dwarf and Ndc80^dwarf are otherwise identical. CH: calponin homology domains; RWD: RWD domains; CC: coiled-coil regions, including residues in junction.

Figure 2

Crystal structures of Ndc80c^dwarf and Ndc80c^e-dwarf. (A) Ndc80c^dwarf. Paired CH domains of Ndc80 and Nuf2 on the left; paired RWD domains on the right; tetramer junction in the middle. R1: region of two-chain coiled coil. R2: region of four-chain overlap. R3: region of three-chain overlap. (B) Ndc80c^e-dwarf. Labels as in (A). Dashed line: Ndc80 residues 250-259, not resolved in electron density map. The structure was determined with a bound, single-chain antibody, omitted for clarity from the figure. See also Figure S2.

Figure 3

Central region of Ndc80c^{dwarf/e-dwarf}. (A) Top: Diagram of Ndc80c^e-dwarf; regions R1, R2 and R3 are identical in the Ndc80c^dwarf and Ndc80^e-dwarf structures (Cα rmsd = 0.8 Å). Middle: Expanded view of R1, R2 and R3. Arrow points to an additional, “stutter” residue in the third heptad in R1. Residues shown as purple sticks (Ndc80: I650 and I647; Spc24: L8 and F14) form a hydrophobic surface that contacts the nanobody (Figure S3). Bottom: cross section diagrams to show two-chain, four-chain, and three-chain packing in R1, R2 and R3, respectively. (B) Expanded view of a network of aromatic and ionic interactions, conserved in fungi. See also Figure S3.

Figure 4

Comparison of yeast and human Ndc80 and Nuf2 CH domains and Ndc80c hinge. (A) Superposition of the Ndc80 CH domain from human (in blue; coordinates from chain B of PDB entry 2VE7) and yeast (in red, chain A of the Ndc80c^dwarf crystal structure). (B) Superposition of Nuf2 CH domains from human (in yellow; chain D of PDB entry 2VE7) and yeast CH domain (in orange; chain B of the Ndc80c^dwarf crystal structure). (C) Structure of CH domains and adjacent coiled coil from Ndc80c^e-dwarf as both secondary-structure ribbons and transparent surface. Side bar marks regions of the structure; red and yellow numbers correspond to Ndc80 and Nuf2, respectively. (D) Structure of Ndc80c^dwarf juxtaposed on transparent surface of Ndc80c^e-dwarf. The arrows show the reorientation of the Ndc80:Nuf2 CH domains with respect to the coiled-coil axis. Structures aligned with respect to residues encompassing the R1 and R2 segments of the middle region with an RMSD of 0.8 Ų. (E) Fulcrum of hinge, showing cluster of aromatic residues. See also Figure S3E and Supplemental Movie 1.