Crystal structure of the yeast inner kinetochore subunit Cep3p

Abstract

In budding yeast, the four-protein CBF3 complex (Skp1p-Ctf13p-Cep3p-Ndc10p) initiates kinetochore assembly by binding to the CDEIII locus of centromeric DNA. A Cep3p dimer recruits a Skp1p-Ctf13p heterodimer and contacts two sites on CDEIII. We report here the crystal structure, determined at 2.8 A resolution by multiple isomorphous replacement with anomalous scattering, of a truncated Cep3p (Cep3p [47-608]), comprising all but an N-terminal, Zn(2)Cys(6)-cluster, DNA-binding module. Cep3p has a well-ordered structure throughout essentially all of its polypeptide chain, unlike most yeast transcription factors, including those with Zn(2)Cys(6) clusters, such as Gal4p. This difference may reflect an underlying functional distinction: whereas any particular transcription factor must adapt to a variety of upstream activating sites, Cep3p scaffolds kinetochore assembly on centromeres uniformly configured on all 16 yeast chromosomes. We have, using the structure of Cep3p (47-608) and the known structures of Zn(2)Cys(6)-cluster domains, modeled the interaction of Cep3p with CDEIII.

Figure 1. Structure of Cep3p

A. Ribbon diagram of Cep3p, colored from blue at the N-terminus (54) to red at the C-terminus (608). Domain 1 (the N-terminal Zn₂Cys₆ cluster), absent in this construct, is located immediately before helix αA. B. Domain 2 (residues 78−229) has a structure similar to the globin fold. C. Domain 3 (residues 230−608) forms a left-handed solenoid composed of seven helical zig-zags which encircles αA. Disordered loops absent from electron density link αM to αN and αU to αV.

Figure 2. The Cep3p dimer

A. Ribbon diagram of the Cep3p dimer viewed perpendicular to the dyad (side view). A ribbon diagram of a monomer in the same orientation, colored as in Figure 1, is provided for reference. B. The Cep3p dimer viewed along the dyad, with domain 3 closest to the viewer (bottom view).

Figure 3. Evolutionary conservation of Cep3p

A. Multiple sequence alignment of Cep3p orthologs from ten point centromere-containing fungi. The alignment is numbered according to the S. cerevisiae Cep3p sequence. Secondary structural elements are indicated above the alignment. Residues participating in dimer contacts are indicated by asterisks above the alignment. Shaded boxes indicate conserved (gray) and invariant (black) residues. B. Side view and bottom view of the Cep3p dimer molecular surface colored by degree of conservation. The dotted ovals indicate two regions of high conservation that may represent Ctf13p interaction surfaces. Residues on the apex are highly variable, and they are presumably not involved in conserved protein-protein contacts.

Figure 4. A model of CBF3-DNA assembly

A. Multiple sequence alignment of the first 27 bases of the 56-bp DNAse I footprint of CDEIII from the 16 S. cerevisiae centromeres. The center of pseudosymmetry at the conserved base 14G is denoted by a diamond. Highly conserved positions are shaded. The positions of Cep3p (green) and Ctf13p (red) protein-DNA crosslinks (Espelin et al., 1997) are indicated by arrows. A B-DNA model of CDEIII is shown in cartoon form, with the putative Cep3p half-sites in green and the putative Ctf13p major groove binding site in red. B. The Zn₂Cys₆ cluster domain. Multiple sequence alignment of Zn₂Cys₆ cluster domains from six S. cerevisiae proteins, with conserved residues shaded and secondary structural elements indicated above the alignment. The length of the linker connecting the Zn₂Cys₆ cluster with αA (in Cep3p) or with the coiled-coil dimerization element (in the other proteins), is indicated, along with the DNA sites recognized by each protein. The Zn₂Cys₆ cluster from Hap1p (PDB ID 1WHT) is presented as a ribbon diagram with the Zn atoms and Cys sidechains shown (King et al., 1999). The two-residue insertion between Cys5 and Cys6 that forms the 3₁₀ helix in Hap1p is also present in Cep3p. C. A model for Cep3p-CDEIII binding. Zn₂Cys₆ clusters from the Hap1p structure have been docked into the two Cep3p half-sites on CDEIII. The twofold axis of Cep3p and the pseudo twofold axis of CDEIII have been superimposed. The polarity of the left-half site is ambiguous; this model approximates what the Cep3p-CDEIII complex could look like if the left half-site forms an inverted repeat (as in Gal4p) retaining overall two-fold symmetry. D. Model of CBF3-CDEIII Assembly. Ctf13p must bind asymmetrically to the Cep3p dimer and contact CDEIII in the major groove halfway between the Cep3p half-sites, as well as link Cep3p to Ndc10p. The Skp1p-Ctf13p heterodimer (purple) has been placed in an arbitrary position that fulfills those constraints. Ndc10p (orange) is modeled to reflect DNA crosslinking and electrophoretic mobility shift data, which suggest that one Ndc10p dimer binds the first 56 bases of CDEIII (as part of the core CBF3 complex), and a second Ndc10p dimer binds the region between 56 and 88 bp (to form the extended CBF3 complex) (Espelin et al., 1997).