Lee S , Thebault P , Freschi L , Beaufils S , Blundell TL , Landry CR , Bolanos-Garcia VM , Elowe S .

GMX-191597 Canadian Institutes of Health Research , RG44650 Wellcome Trust

	FIGURE 1. Identification of a TPR motif in the N-terminal region of Mps1. A, amino acid sequence alignment of human N-terminal Mps1 and BUB1 and BUBR1 from different species. Secondary structure elements are mapped onto the crystal structure of N-terminal BUBR1 (Protein Data Bank code 2WVI). Figure was generated with ESPript (68). B, structure model of N-terminal Mps1 predicts this region is organized as a triple tandem of the TPR motif. C, model highlighting the Mps1 residues that are conserved and located in positions that define a canonical TPR motif. Most of these conserved residues are predicted to be engaged in stabilizing stacking interactions. Purple indicates the phosphorylation site serine 80 is highlighted. D, far-UV CD confirms N-terminal Mps1 is organized as a predominantly α-helical region. Inset, the thermal denaturation of this domain is highly cooperative and follows a two-state unfolding process. E, size-exclusion chromatogram of molecular mass markers only (●): peak 1, bovine serum albumin; peak 2, ovalbumin; peak 3, chymotrypsinogen A; peak 4, ribonuclease A. For the second chromatogram (−), the same molecular mass markers were combined with Mps1(1–239) prior to gel filtration. Mps1(1–239) retention time was closed to that of ovalbumin (43 kDa) thus revealing the former self-associates to form stable dimers.
	FIGURE 2. Biophysical characterization of the N-terminal fragment Mps1(1–239). Interfacial properties of Mps1(1–239), TPR Bub1, and TPR BubR1 are shown. Surface pressure (A) and surface concentration (Γ = 0.2 Δ) (B) together with the corresponding ellipsometric angle at air/water interfaces were determined by null ellipsometry measurements. Solutions of Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●) are shown. Protein solutions were prepared at 30 μg/ml in 20 mm phosphate buffer, pH 7. C, surface pressure versus surface concentration of Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●). θ corresponds to the slope dπ/dΓ, and Γ0 is the surface concentration at which the surface pressure becomes different from zero. Γ0 is calculated from the intersect of the slope π versus Γ. Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●) at 1 μg/ml in 20 mm phosphate buffer, pH 7, are shown. D, rheology measurements of Mps1(1–239) (□), TPR Bub1 (○), and TPR BubR1 (●). The graph shows the evolution of the shear elastic constant, μ, versus time measured at the fixed frequency of 5 Hz, during protein adsorption at the interface. Protein solutions were prepared at 30 μg/ml in 20 mm phosphate buffer, pH 7. The error bar on μ is ± 5 mN/m. Inset, at the end of the kinetic (around 9 h, indicated by the arrow in the graph) the angular deviation θ(ω) versus the pulsation was measured. The curves correspond to Mps1(1–239). An elastic layer model (harmonic oscillator) fit the imaginary and real part of the response. For clarity, the imaginary part has been plotted versus −ω.
	FIGURE 3. Mps1 TPR domain appeared after the emergence of deuterostomes. A, human Mps1 overlapping fragments (length, 160 amino acids; offset, 10 amino acids) were blasted (PSI-BLAST) against the entire NCBI sequence database (nonredundant). For each listed organism for which we could find a hit for one or more fragments, we show if the hit was highly significant (red rectangles, E-value <1e-5), close to the threshold (orange rectangles, ∼1e-5 < E-value < 1), or not significant (yellow rectangles, E-value ≥1). If we could not find a hit for a specific fragment, the corresponding rectangle is gray. The column highlighted between white lines corresponds to the exact hMps1 TPR region. B, secondary structure prediction of the TPR region of hMps1 and the corresponding region in Mps1 orthologues. Alignment and prediction were performed using PSIPRED (33).
	FIGURE 4. Localization of Mps1(1–239). A, localization of 3×MYC-Mps1 constructs. HeLa cells were transfected with 3×MYC-Mps1 WT (row i), -Mps1-KD (row ii) or Mps1(1–239) (rows iii and iv) and synchronized in mitosis by release from a single thymidine arrest before being fixed and stained for immunofluorescence with antibodies against MYC (green) and CREST (blue). The panel on the right shows the colocalization profile between MYC and CREST signal across sister kinetochore pairs (3–4 pairs). A, row iii, colocalization of one sister kinetochore pair is included, and the pair is detailed in the inset. B, immunofluorescence of cells expressing mCherry alone (row i, medium; row iii, highly overexpressing) or mCherry-Mps1(1–239) (row ii, medium; row iv, highly overexpressing) were synchronized in mitosis and fixed for immunofluorescence as in A. Cells were stained with anti-Mps1 antibodies (green) and CREST autoimmune serum (blue). C, endogenous Mps1/CREST signal ratio (arbitrary units; a.u.) in cells highly overexpressing mCherry or mCherry-Mps1(1–239), n = 9 cells each. D, cells were transfected, synchronized, and fixed as in A and were then stained for the indicated proteins. Kinetochore localization was scored for these proteins in cells overexpressing Mps1(1–239) relative to control cells.
	FIGURE 5. Functional analysis of Mps1(1–239). A, HeLa cells were transfected with mCherry or mCherry-Mps1A and then synchronized in STLC followed by release into MG132 according to the schematic. After 1.5 h of MG132 treatment, cells were fixed and processed for immunofluorescence with antibodies against α-tubulin (green) and CREST (Cy5, illustrated here in magenta). Hoechst 33342 was used to visualize the DNA (blue). Chromosome alignment at metaphase plates was examined in cells expressing either mCherry or mCherry-Mps1(1–239) and grouped into the three indicated categories based on the extent of the alignment. Only cells in which both spindle poles were visible in the same plane were counted (n = 3, ≥100 cells/experiment). B, cells transfected with either mCherry or mCherry-Mps1 were treated for 16 h either with DMSO as control or 3.3 μm nocodazole (Noco), fixed, and stained with Hoechst 33342 to visualize the DNA. The mitotic index of transfected cells was counted (n = 5, ≥100 cells/experiment). C, cells were transfected as in B but were also simultaneously transfected with siRNA oligonucleotides targeting Gl2 (as control) or CenpE. 48 h after transfection, the cells were fixed and stained as in B before mitotic index counts were performed (n = 3, ≥100 cells/experiment). D, cells were transfected with either mCherry or mCherry-Mps1(1–239) and treated with 3.3 μm nocodazole for 16 h before being fixed and stained for Mad2 (green) and CREST (Cy5, shown here in blue). E, quantification of the Mad2/CREST ratio from cells in D. F, 293T cells were transfected with 3×MYC Mps1-WT or 3×MYC Mps1ΔN and treated with nocodazole for 16 h before being harvested. Immunoprecipitated Mps1 fragments were resolved by SDS-PAGE and immunoblotted with anti-Thr(P)-676, anti-Thr(P)-686, and anti-Ser(P)-S821 Mps1 antibodies. The membranes were stripped and reprobed for MYC to demonstrate equal input.
	FIGURE 6. Model for Mps1 TPR function. A, Mps1-WT can both dimerize and dock at KTs, thus increasing its local concentration and activity. Mps1 autophosphorylation or phosphorylation of a KT protein allows for its release into the cytoplasm. B, inactive Mps1 can dimerize and localize but cannot phosphorylate itself or other targets and is thus not released from the KT as efficiently as the WT protein. C, Mps1 lacking a functional TPR cannot localize to the KT and cannot dimerize resulting in reduced concentration of Mps1 protein and reduced kinase activity. This is manifest as reduced autophosphorylation and reduced phosphorylation of potential targets. Ultimately this results in suboptimal Mps1 activity.

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