Phosphorylated T866 decreases binding of Stu2 to Ndc80c. (A) The crystal structure of Stu2 C-terminus bound to Ndc80^Dwarf (PDB 7KDF); see Zahm et al. (2021). Ndc80c^Dwarf in grey, Stu2 C-terminus in blue. (B) Zoom in of crystal structure showing hydrophobicity of Stu2 and Ndc80c amino acids rendered using ChimeraX on the Kyte–Doolittle scale (kdHydrophobicity). Ndc80c proteins in surface representation; Stu2 in ribbon representation. Left: Unmodified Stu2^T866 as in PDB 7KDF. Right: Modeling of Stu2^pT866. (C) Schematic representation of fluorescence polarization competition assay. Oregon Green–labeled Stu2 CTS peptide prebound to Ndc80^Dwarf was then incubated with unlabeled Stu2 peptide with or without pT866, and fluorescence polarization was measured. (D) A Stu2 CTS peptide (855–888), a Stu2 CTS peptide containing a phosphothreonine at position 866, and a peptide with a randomized CTS sequence were subjected to serial twofold dilutions, and each dilution was mixed 1:1 with a solution containing 30 µM Ndc80^Dwarf and 200 nM Oregon Green–labeled CTS peptide, also in resuspension buffer. Fluorescence polarization was measured in triplicate in 96-well plates. Error bars are SD. Kd calculated with four parameter logistic curve fitting in Prism. (E) Exponentially growing stu2-AID cultures expressing an ectopic copy of STU2 (STU2^WT, M622; stu2^T866E, M1448; stu2^T866V, M4398) as well as DSN1-6His-3Flag at the genomic locus were treated with auxin 30 min prior to harvesting. Kinetochore particles were purified from lysates by anti-Flag immunoprecipitation (IP) and analyzed by immunoblotting. (F) Exponentially growing stu2-AID pMET-CDC20 cultures with an ectopically expressed STU2-GFP allele (STU2^WT-GFP, M2599; stu2^T866E-GFP, M2600; and stu2^T866V-GFP, M4447) that also contained SPC110-mCherry (spindle pole) were shifted to auxin-containing media to degrade Stu2-AID and supplemented with methionine to arrest cells in metaphase by depleting Cdc20. Cells were fixed and imaged to determine kinetochore-proximal Stu2-GFP. Bars represent the mean of n = 96–111 individual measurements. Error bars are SEM. P values from two-tailed unpaired t tests. Source data are available for this figure: SourceData F2.