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. 2008 Jan;9(1):56-62.
doi: 10.1038/sj.embor.7401139. Epub 2007 Dec 7.

Insights into kinetochore-DNA interactions from the structure of Cep3Delta

Alan Purvis  1 , Martin R Singleton
Affiliations

Insights into kinetochore-DNA interactions from the structure of Cep3Delta

Alan Purvis et al. EMBO Rep. 2008 Jan.

Abstract

The CBF3 complex is an essential core component of the budding yeast kinetochore and is required for the centromeric localization of all other kinetochore proteins. We determined the crystal structure of a large section of the protein Cep3 from CBF3, which is the only component with obvious DNA-binding motifs. The protein adopts a roughly bilobal shape, with an extended dimerization interface. The dimer has a large central channel that is sufficient to accommodate duplex B-form DNA. The zinc-finger domains emerge at the edges of the channel, and could bind to the DNA in a pseudo-symmetrical manner at degenerate half-sites in the centromeric sequence. We propose a mechanism for the modulation of DNA affinity by an acidic activator domain, which could be applicable to a wider family of transcription factors.

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Figures

Figure 1
Figure 1
Structure of the budding yeast centromere and organization of the CBF3 complex. (A) Schematic diagram of the budding yeast centromere element showing the location and scale of the CDE elements, as well as the consensus DNA composition in CDEIII. (B) Diagram of the four components of the CBF3 complex on DNA. (C) Schematic of the Cep3 sequence against Gal4 showing the locations of the conserved DNA-binding domain (yellow) and acidic patch (purple). The orange bar indicates the fragment described in this study. (D) Sequence alignment of members of the zinc-cluster domains from members of the Gal4-family of transcription factors against the amino-terminal of Cep3. The secondary structure elements are shown above the alignment, and totally conserved residues are shown in red.
Figure 2
Figure 2
Crystal structure of the Cep3Δ monomer and dimerization in solution. (A) Ribbon diagram of the Cep3Δ monomer with domain 1 shown in pink and domain 2 in blue. (B) Superimposition of domain 2 of Cep3 (shown in orange) against three HEAT repeats from β-importin (Protein Data Bank i.d. 1qgk; shown in grey). (C) Gel filtration profiles of full-length Cep3 and Cep3Δ on a calibrated column. The molecular weight standards are indicated above the trace.
Figure 3
Figure 3
Structure of the Cep3Δ dimer and electrostatic properties. (A) The proposed biological dimer, formed around a crystallographic twofold axis. A space filling view, rotated by 90° shows the groove on the surface. (B) Electrostatic surface representations of the dimer on the concave (left) and convex (right) surfaces.
Figure 4
Figure 4
Cep3–DNA interactions. (A) Proposed model for DNA binding. An ideal B-form duplex has been placed in the cleft, with the triplet half-sites (green) equidistant from the amino-terminal of the protein. The potential location of the linkers and zinc clusters are shown schematically. (B) Model of how Cep3 and Ctf13 could simultaneously bind to and encircle the duplex. (C) The 33 base pair DNA sequences used for binding studies. All four unlabelled DNAs were used as competitors against the FAM6 fluorescently labelled native CDEIII probe. Mutations of the native CDEIII sequence are italicised and restricted to the proposed Cep3 binding sites. (D) Electrophoretic mobility-shift assay (EMSA) performed with 4.2 μM fluorescent probe with increasing amounts (4.2, 8.3, 12.7 μM) of Cep3Δ (lanes 2–4), Cep31−608 (lanes 5–7), or 12.7 μM Cep31−608 with increasing amounts (3, 6, 12 × probe concentration) native CDEIII competitor (lanes 8–10). (E) EMSA performed with 4.2 μM fluorescent probe and 12.7 μM Cep31−608 (lane 1) with increasing amounts (3, 6, 12 × probe concentration) of site 2 mutant competitor (lane 2–4), sites 1 mutant competitor (lane 5–7), or sites 1 and 2 mutant competitor (lanes 8–10).
Figure 5
Figure 5
Proposed function for carboxy-terminal acidic motif. (A) The acidic patch in the amino-terminal of Cep3Δ forms a disordered loop (dotted purple line). The N-terminal of the protein is coloured pink and the approximate location of the zinc cluster (ZC) indicated. (B) The acidic loop might directly contact the zinc cluster in response to binding by other factors and modulate target affinity.
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