Recognition of the centromere-specific histone Cse4 by the chaperone Scm3

Abstract

A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)(2) heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.

Fig. 1.

Scm3 as a Cse4-specific chaperone. (A) Polycistronic expression of K. lactis Cse4 and H4 in the presence or absence of Scm3. Scm3 has tandem N-terminal His₆ tag and MBP tag; Cse4 and H4 have N-terminal His₆ tag. S, soluble fraction; I, insoluble fraction; Ni, Ni-NTA eluate; MBP, amylose eluate. The soluble fraction of cell lysates was applied to a Ni-NTA affinity column, and the eluate was then applied to an amylose affinity column. The Ni-NTA eluate contains all soluble, His-tagged proteins; the amylose eluate identifies proteins that form stable complexes with MBP–Scm3. (B) Polycistronic expression of K. lactis H3 and H4 in the presence or absence of Scm3.

Fig. 2.

Structure of the Scm3:Cse4:H4 heterotrimer. (A) Ribbon diagram of the Scm3:Cse4:H4 heterotrimer. Residues 44–103 of Scm3, residues 108–180 of Cse4, and residues 24–97 of H4 are in red, dark blue, and cyan, respectively. (B) Sequence conservation in Scm3 and Cse4. Colors ramped from red and blue, respectively, to yellow, corresponding to degree of conservation in multiple sequence alignments (Fig. S4 A and B) of Scm3 and Cse4 from K. lactis, S. cerevisiae, Schizosaccharomyces pombe, bovine, mouse, and human. (C) Some key interacting residues of Cse4 and Scm3. (D) Amylose pull-down with MBP–Scm3 (the bait) coexpressed with H4 and mutants of Cse4. Mutants of Cse4 with substitutions at various positions of their H3 counterparts were coexpressed with MBP–Scm3 and H4. Substitutions in Cse4 loop1 are residues 76–88 of H3 (AQDFKTDLRFQSS) for residues 150–165 of Cse4 (TDQFTTESEPLRWQSM). The volume of sample used for SDS-PAGE was adjusted based on the amount of MBP–Scm3.

Fig. 3.

Structure of the (Cse4 : H4)₂ heterotetramer and structural alignment with the Scm3:Cse4:H4 heterotrimer. (A) Ribbon diagram of the (Cse4 : H4)₂ heterotetramer. Cse4 is in dark green; H4 is in light green. (B) Superposition of the Scm3:Cse4:H4 and (Cse4 : H4)₂ heterotetramers. The structures of Cse4 and H4 in the heterotrimer align well with those in the heterotetramer. In the superposition, the central helix of Scm3 clashes with helix III of neighboring Cse4 in the tetramer (red dashed circle), illustrating that Scm3 binding and formation of a Cse4:H4 tetramer are mutually exclusive.

Fig. 4.

Schematic model for Cse4:H4 incorporation at budding-yeast centromeres. Model for Cse4 nucleosome assembly. Scm3 stabilizes the Cse4:H4 heterodimer in solution by forming an Scm3:Cse4:H4 heterotrimeric complex, which is localized to a centromere through an interaction of Scm3 with Ndc10 (8). An Ndc10 dimer, as a part of the CBF3 complex, recognizes and associates with centromeric DNA and generates a DNA loop (U-S.C. and S.C.H., manuscript in preparation). Tetramer formation and DNA association detach Scm3 from Cse4:H4, but Scm3 remains at the centromere through its contact with Ndc10.