Figure 6.
Interactions between the Mis12cMtw1chead 1 and head 2 domains. (A) Observed CL-MS crosslinks on the KHB-RWDM complex, visualized using the molecular model from Fig. S5 B. Inter-subunit crosslinks between residues in the Mis12cMtw1c head 1 and head 2 domains are highlighted, and relevant Cα atoms of crosslinked residues are shown as spheres. Crosslinks shown are between residues that satisfy the physical distance constraint of the chemical crosslinker. Crosslinks between two residues are only displayed if both residues are visible in the panel. (B) Cryo-EM density map with improved occupancy for the Mis12cMtw1c head 2 domain (Fig. S4), interpreted and color-coded according to the experimental model of ScMis12cMtw1c. (C) Experimental cryo-EM model of ScMis12cMtw1c head 1 and head 2 domains with Dsn1 auto-inhibitory region (Dsn1AI) at the base of the ScMis12cMtw1c stalk domain. (D) CL-MS crosslinks from the ScMis12cMtw1 dataset that satisfy the physical distance constraint of the chemical crosslinker, indicated in green, mapped onto ScMis12cMtw1c head 1, head 2, and stalk domains and Dsn1AI. (E) Molecular model showing the interaction between the ScMis12cMtw1c head 1 domain and Dsn1AI (residues 229–255). ScMis12cMtw1c head 2 domain is hidden. (F) Multiple sequence alignment of Dsn1 protein from distantly related budding yeast demonstrates that the KlDsn1201-230 segment of the Dsn1 N-IDR is conserved. (G) ITC experiments to determine the identity of the unassigned α-helical-like density. (i) Titration of Dsn1228-254 peptide to Knl1c:Mis12cMtw1c complex (KHB-RWDMDsn1Δ257) with Knl1Spc105 residues 1–444 and Dsn1 residues 1–257 deleted. (ii) Titration of Dsn1228-254 peptide to a head 1–deleted Knl1c:Mis12cMtw1c complex (KHB-RWDMDsn1Δ257_Δhead1): Knl1Spc105 residues 1–444, Dsn1 residues 1–257, Mis12Mtw1 residues 1–106, and Nnf1 residues 1–104 deleted. (iii) Titration of Dsn1228-254 peptide to KHB-RWDMDsn1Δ257 complex (KHB-RWDMDsn1Δ257_Mis12-3A) containing mutant Mis12Mtw1_E69A,E73A,E77A. (iv) ITC data for phosphomimetic mutations at Aurora B sites in Dsn1228-254. Titration of Dsn1228–254 peptide with S240E and S250E phosphomimetic mutations (Dsn1228-254,S240E,S250E) to Knl1c:Mis12cMtw1c complex (KHB-RWDMDsn1Δ257) with Knl1Spc105 residues 1–444 and Dsn1 residues 1–257 deleted. KD and n values are an average from three i) and two ii), iii), and iv) experiments (technical repeats). Refer to the image caption for details. Panel A shows C L-M S crosslinks mapped between M i s 12 c M t w 1 c head 1 and head 2 domains, highlighting residue interactions within distance constraints. Panel B shows cryo-E M density map of M i s 12 c M t w 1 c with improved head 2 occupancy, fitted with structural model. Panel C shows experimental model of M i s 12 c M t w 1 c highlighting head 1 and head 2 arrangement and D s n 1 auto-inhibitory region. Panel D shows mapped crosslinks across head, stalk, and D s n 1 A I regions validating structural proximity. Panel E shows molecular interaction between head 1 domain and D s n 1 auto-inhibitory segment (residues 229 to 255). Panel F shows sequence alignment of D s n 1 highlighting conserved N-terminal region across species. Panel G shows I T C binding assays quantifying interaction of D s n 1 peptides with M i s 12 c M t w 1 c complexes under different conditions.

Interactions between the Mis12c Mtw1c head 1 and head 2 domains. (A) Observed CL-MS crosslinks on the KHB-RWDM complex, visualized using the molecular model from Fig. S5 B. Inter-subunit crosslinks between residues in the Mis12cMtw1c head 1 and head 2 domains are highlighted, and relevant Cα atoms of crosslinked residues are shown as spheres. Crosslinks shown are between residues that satisfy the physical distance constraint of the chemical crosslinker. Crosslinks between two residues are only displayed if both residues are visible in the panel. (B) Cryo-EM density map with improved occupancy for the Mis12cMtw1c head 2 domain (Fig. S4), interpreted and color-coded according to the experimental model of ScMis12cMtw1c. (C) Experimental cryo-EM model of ScMis12cMtw1c head 1 and head 2 domains with Dsn1 auto-inhibitory region (Dsn1AI) at the base of the ScMis12cMtw1c stalk domain. (D) CL-MS crosslinks from the ScMis12cMtw1 dataset that satisfy the physical distance constraint of the chemical crosslinker, indicated in green, mapped onto ScMis12cMtw1c head 1, head 2, and stalk domains and Dsn1AI. (E) Molecular model showing the interaction between the ScMis12cMtw1c head 1 domain and Dsn1AI (residues 229–255). ScMis12cMtw1c head 2 domain is hidden. (F) Multiple sequence alignment of Dsn1 protein from distantly related budding yeast demonstrates that the KlDsn1201-230 segment of the Dsn1 N-IDR is conserved. (G) ITC experiments to determine the identity of the unassigned α-helical-like density. (i) Titration of Dsn1228-254 peptide to Knl1c:Mis12cMtw1c complex (KHB-RWDMDsn1Δ257) with Knl1Spc105 residues 1–444 and Dsn1 residues 1–257 deleted. (ii) Titration of Dsn1228-254 peptide to a head 1–deleted Knl1c:Mis12cMtw1c complex (KHB-RWDMDsn1Δ257_Δhead1): Knl1Spc105 residues 1–444, Dsn1 residues 1–257, Mis12Mtw1 residues 1–106, and Nnf1 residues 1–104 deleted. (iii) Titration of Dsn1228-254 peptide to KHB-RWDMDsn1Δ257 complex (KHB-RWDMDsn1Δ257_Mis12-3A) containing mutant Mis12Mtw1_E69A,E73A,E77A. (iv) ITC data for phosphomimetic mutations at Aurora B sites in Dsn1228-254. Titration of Dsn1228–254 peptide with S240E and S250E phosphomimetic mutations (Dsn1228-254,S240E,S250E) to Knl1c:Mis12cMtw1c complex (KHB-RWDMDsn1Δ257) with Knl1Spc105 residues 1–444 and Dsn1 residues 1–257 deleted. KD and n values are an average from three i) and two ii), iii), and iv) experiments (technical repeats).

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