Figure 7.
Regulation of Mis12cMtw1cauto-inhibition by Aurora B kinase and its effect on the interaction with CENP-CMif2. (A) Structural alignment of the crystal structure of K. lactis CENP-CMif2 residues 4–36 (KlCENP-CMif2_4-36) as bound to the KlMis12cMtw1c head 1 domain (PDB: 5T59) (Dimitrova et al., 2016) to the experimental model of ScMis12cMtw1c (this study). The models are colored by chain; Mis12Mtw1 and Nnf1 are colored identically in 5T59 and our experimentally determined model. KlCENP-CMif2_4-36 is shown in space filling representation to highlight lack of steric clash, and overlapping binding site, with Dsn1AI. Only KlCENP-CMif2_4-36 is shown. (B) SEC elution chromatograms of attempts to reconstitute the interaction between Knl1c:Mis12cMtw1c complexes containing different Dsn1 N-terminal truncation and/or phosphomimetic mutants and CENP-CMif2 residues 1–63 (CENP-CMif2_1-63) indicated within red boxes. (C) Coomassie brilliant blue–stained SDS-PAGE gels of the chromatograms shown in B. (B and C) KHB-RWDM = full-length Dsn1. KHB-RWDM2E = full-length Dsn1 containing Dsn1S240E and Dsn1S250E phosphomimetic mutations. KHB-RWDMDsn1Δ257 = Dsn1 residues 1–257 are deleted (Dsn1Δ1-257). KHB-RWDMDsn1Δ226 = Dsn1 residues 1–226 are deleted (Dsn1Δ1-226). KHB-RWDMDsn1Δ226-2E = Dsn1 residues 1–226 are deleted and Dsn1S240E and Dsn1S250E phosphomimetic mutations (Dsn1Δ1-226,S240E,S250E). CENP-CMif2_1-63 is indicated within a red box. (D) ITC data for (i) CENP-CMif2_1-38 interactions with KM, and (ii) CENP-CMif2_1-38 interactions with KM2E. KD and n values are an average from three experiments (technical repeats). Source data are available for this figure: SourceData F7. Refer to the image caption for details. Panel A shows structural alignment of C E N P-C M i f 2 peptide with M i s 12 c M t w 1 c head 1, highlighting overlap with D s n 1 auto-inhibitory region. Panel B shows S E C elution profiles comparing binding of C E N P-C M i f 2 to different M i s 12 c M t w 1 c mutant complexes. Panel C shows S D S-PAGE gels validating protein composition across S E C fractions for each condition. Panel D shows I T C binding assays measuring interaction between C E N P-C M i f 2 peptide and M i s 12 c M t w 1 c complexes, including phosphomimetic mutants.

Regulation of Mis12c Mtw1c auto-inhibition by Aurora B kinase and its effect on the interaction with CENP-C Mif2 . (A) Structural alignment of the crystal structure of K. lactis CENP-CMif2 residues 4–36 (KlCENP-CMif2_4-36) as bound to the KlMis12cMtw1c head 1 domain (PDB: 5T59) (Dimitrova et al., 2016) to the experimental model of ScMis12cMtw1c (this study). The models are colored by chain; Mis12Mtw1 and Nnf1 are colored identically in 5T59 and our experimentally determined model. KlCENP-CMif2_4-36 is shown in space filling representation to highlight lack of steric clash, and overlapping binding site, with Dsn1AI. Only KlCENP-CMif2_4-36 is shown. (B) SEC elution chromatograms of attempts to reconstitute the interaction between Knl1c:Mis12cMtw1c complexes containing different Dsn1 N-terminal truncation and/or phosphomimetic mutants and CENP-CMif2 residues 1–63 (CENP-CMif2_1-63) indicated within red boxes. (C) Coomassie brilliant blue–stained SDS-PAGE gels of the chromatograms shown in B. (B and C) KHB-RWDM = full-length Dsn1. KHB-RWDM2E = full-length Dsn1 containing Dsn1S240E and Dsn1S250E phosphomimetic mutations. KHB-RWDMDsn1Δ257 = Dsn1 residues 1–257 are deleted (Dsn1Δ1-257). KHB-RWDMDsn1Δ226 = Dsn1 residues 1–226 are deleted (Dsn1Δ1-226). KHB-RWDMDsn1Δ226-2E = Dsn1 residues 1–226 are deleted and Dsn1S240E and Dsn1S250E phosphomimetic mutations (Dsn1Δ1-226,S240E,S250E). CENP-CMif2_1-63 is indicated within a red box. (D) ITC data for (i) CENP-CMif2_1-38 interactions with KM, and (ii) CENP-CMif2_1-38 interactions with KM2E. KD and n values are an average from three experiments (technical repeats). Source data are available for this figure: SourceData F7.

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