Structural basis for recognition of centromere histone variant CenH3 by the chaperone Scm3

Abstract

The centromere is a unique chromosomal locus that ensures accurate segregation of chromosomes during cell division by directing the assembly of a multiprotein complex, the kinetochore. The centromere is marked by a conserved variant of conventional histone H3 termed CenH3 or CENP-A (ref. 2). A conserved motif of CenH3, the CATD, defined by loop 1 and helix 2 of the histone fold, is necessary and sufficient for specifying centromere functions of CenH3 (refs 3, 4). The structural basis of this specification is of particular interest. Yeast Scm3 and human HJURP are conserved non-histone proteins that interact physically with the (CenH3-H4)(2) heterotetramer and are required for the deposition of CenH3 at centromeres in vivo. Here we have elucidated the structural basis for recognition of budding yeast (Saccharomyces cerevisiae) CenH3 (called Cse4) by Scm3. We solved the structure of the Cse4-binding domain (CBD) of Scm3 in complex with Cse4 and H4 in a single chain model. An α-helix and an irregular loop at the conserved amino terminus and a shorter α-helix at the carboxy terminus of Scm3(CBD) wraps around the Cse4-H4 dimer. Four Cse4-specific residues in the N-terminal region of helix 2 are sufficient for specific recognition by conserved and functionally important residues in the N-terminal helix of Scm3 through formation of a hydrophobic cluster. Scm3(CBD) induces major conformational changes and sterically occludes DNA-binding sites in the structure of Cse4 and H4. These findings have implications for the assembly and architecture of the centromeric nucleosome.

Figure 1. Overall structure and dynamics of scSCH

a, The amino acid sequence and secondary structures of the Cse4-binding domain of Scm3 in scSCH. Also shown are the conserved regions in the Scm3 of S. pombe and human HJURP. The highly conserved residues are highlighted in red. The region in the folded core is shown in dark magenta (see e). b–d, Front, bottom, and back view of the scSCH structure shown in ribbon. Scm3, Cse4, and H4 are in magenta, cyan, and dark green, respectively. The full sequence of scSCH is M-His₆-KK-Cse4_150–227-LVPRGS-Scm3_93–169-GDK-H4_42–103.

Figure 2. The N-terminal region (181–190) of the α2 helix of Cse4 is the Scm3 recognition motif

a, Ile110, Ile111, Tyr114, and Ile117 (balls in magenta) in the αN helix of Scm3 form a hydrophobic cluster with Cse4-specific residues Met181 and Met184 (balls in cyan) in the α2 helix of Cse4. b, Trp107 (balls in magenta) in the αN helix of Scm3 has close interactions with the Cse4-specific residue Ala189 (balls in cyan) in the α2 helix of Cse4. Ser190 is also a Cse4-specific residue (balls in cyan). c, SDS-page gels showing the pull-down results with mutants of H3. Upper panel shows the input of H3 mutants and H4. Lower panel shows the molecules eluted from His6-Scm3 (Scm3_65–169)-bound beads with 250 mM imidazole. d, Illustration of the secondary structures in Cse4 and the Scm3 recognition motif (dark cyan), CATD, and the mutants used in the pull-down experiments. The red squares indicate the four residues that are sufficient for specific recognition of Cse4 by Scm3. The sequences swapped from Cse4 to H3 in the mutations are shown. The sequences that are not changed in the swap are omitted.

Figure 3. Altered interactions in the CATD region in scSCH

a, The region of the α2 helices of Cse4 and H4 in scSCH, showing that there is little side chain interaction between the two helices. b, The corresponding region of the α2 helices of Cse4 and H4 in the Cse4-H4 dimer structure obtained by homology modeling based on the structures of H3 and H4 in the nucleosome, showing that there are many hydrophobic interactions. c, The region of the α2 helices of H3 and H4 in the nucleosome. d, Region of loop 1 of Cse4 and the loop 2 of H4. The side chains of the hydrophobic residues are shown in stick and orange. The extra three residues in loop 1 of Cse4 are shown in stick. e, The corresponding loop 1 of H3 and loop 2 of H4 in the nucleosome structure. The hydrophobic residues are shown in stick and orange.

Figure 4. Scm3 induces large conformational changes in Cse4 and H4 and prevents loop 2 of H4 from binding to DNA

a, Cse4-H4 in scSCH. The extended α3 helix in H4 is shown in light green. The loop of Scm3 pushes loop 1 of Cse4 away from loop 2 of H4 and prevents loop 2 of H4 from binding to DNA. DNA is modeled to bind the loop 2 region of H4 based on the canonical nucleosome structure. b, CENP-A-H4 in the (CENP-A-H4)₂ tetramer. DNA is modeled to bind to loop 2 region of H4 based on the canonical nucleosome struture.