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Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

Lee S, Thebault P, Freschi L, Beaufils S, Blundell TL, Landry CR, Bolanos-Garcia VM, Elowe S - J. Biol. Chem. (2011)

Bottom Line: Deletions within this region result in checkpoint failure and chromosome segregation defects.Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms.Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.

ABSTRACT
Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

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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.
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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.

Mentions: The observation that the multidomain protein kinases Bub1, BubR1, and Mps1 require kinetochore attachment to perform efficiently their essential roles in the SAC prompted us to investigate to what extent the similarity of functions is the result of evolutionary conservation among these checkpoint kinases. Sequence alignments of N-terminal Mps1 (residues 61–210) against Bub1 and BubR1 from various species indicate that the highest similarity of N-terminal Mps1 is with the N-terminal region of BubR1 (20.3% sequence identity, Fig. 1A) followed by N-terminal Bub1 (14.2% sequence identity). Analysis using FUGUE software shows a Z-score for N-terminal Mps1 of 24.36 thus indicating (with 99% confidence) that an evolutionary relationship and common fold exist between the TPR domains of Bub1 and BubR1 and N-terminal Mps1. Because of the higher amino acid sequence identity between human Mps1 and human BubR1, we modeled the N-terminal region of Mps1 using the crystal structure of human BubR1 (38) as template (Fig. 1, B and C). Some conserved features of a canonical TPR can be recognized in the Mps1 structure model, including a pattern of large residues (WYFL) in TPR helix A and smaller ones (ESAG) in TPR helix B at equivalent positions to those described for canonical TPRs (Fig. 1B). Such residue distribution is expected to be important for the establishment of interactions between TPR-forming helices, as these interactions should confer stability to the N-terminal domain (Fig. 1C). Furthermore, the model of the three-dimensional structure includes an additional α-helix immediately downstream of the third TPR unit of N-terminal Mps1, which might function as a C-terminal “capping” helix, as observed in certain TPR structures (39, 40). The model allows the mapping of residues that are fully conserved and predicted to be exposed at the surface. For instance, serine 80, a residue that can be phosphorylated in vivo (41), is mapped onto the flexible loop region that links helices A and B of TPR1 (Fig. 1C). The predicted spatial location of this residue is consistent with its accessibility to protein kinases. These observations prompted us to investigate further the predicted structural similarity between N-terminal Mps1, Bub1, and BubR1 using a multidisciplinary experimental strategy involving biochemical, biophysical, and cellular methods.


Characterization of spindle checkpoint kinase Mps1 reveals domain with functional and structural similarities to tetratricopeptide repeat motifs of Bub1 and BubR1 checkpoint kinases.

Lee S, Thebault P, Freschi L, Beaufils S, Blundell TL, Landry CR, Bolanos-Garcia VM, Elowe S - J. Biol. Chem. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3285366&req=5

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.
Mentions: The observation that the multidomain protein kinases Bub1, BubR1, and Mps1 require kinetochore attachment to perform efficiently their essential roles in the SAC prompted us to investigate to what extent the similarity of functions is the result of evolutionary conservation among these checkpoint kinases. Sequence alignments of N-terminal Mps1 (residues 61–210) against Bub1 and BubR1 from various species indicate that the highest similarity of N-terminal Mps1 is with the N-terminal region of BubR1 (20.3% sequence identity, Fig. 1A) followed by N-terminal Bub1 (14.2% sequence identity). Analysis using FUGUE software shows a Z-score for N-terminal Mps1 of 24.36 thus indicating (with 99% confidence) that an evolutionary relationship and common fold exist between the TPR domains of Bub1 and BubR1 and N-terminal Mps1. Because of the higher amino acid sequence identity between human Mps1 and human BubR1, we modeled the N-terminal region of Mps1 using the crystal structure of human BubR1 (38) as template (Fig. 1, B and C). Some conserved features of a canonical TPR can be recognized in the Mps1 structure model, including a pattern of large residues (WYFL) in TPR helix A and smaller ones (ESAG) in TPR helix B at equivalent positions to those described for canonical TPRs (Fig. 1B). Such residue distribution is expected to be important for the establishment of interactions between TPR-forming helices, as these interactions should confer stability to the N-terminal domain (Fig. 1C). Furthermore, the model of the three-dimensional structure includes an additional α-helix immediately downstream of the third TPR unit of N-terminal Mps1, which might function as a C-terminal “capping” helix, as observed in certain TPR structures (39, 40). The model allows the mapping of residues that are fully conserved and predicted to be exposed at the surface. For instance, serine 80, a residue that can be phosphorylated in vivo (41), is mapped onto the flexible loop region that links helices A and B of TPR1 (Fig. 1C). The predicted spatial location of this residue is consistent with its accessibility to protein kinases. These observations prompted us to investigate further the predicted structural similarity between N-terminal Mps1, Bub1, and BubR1 using a multidisciplinary experimental strategy involving biochemical, biophysical, and cellular methods.

Bottom Line: Deletions within this region result in checkpoint failure and chromosome segregation defects.Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms.Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.

ABSTRACT
Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region.

Show MeSH
Related in: MedlinePlus