Structural analysis reveals features of the spindle checkpoint kinase Bub1-kinetochore subunit Knl1 interaction

J Cell Biol. 2012 Feb 20;196(4):451-67. doi: 10.1083/jcb.201110013. Epub 2012 Feb 13.

Abstract

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR-KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Blotting, Western
  • Crystallization
  • Fluorescent Antibody Technique
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Kinetochores / physiology*
  • Microtubule-Associated Proteins / chemistry*
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Mitosis / physiology
  • Molecular Sequence Data
  • Nuclear Pore Complex Proteins / genetics
  • Nuclear Pore Complex Proteins / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Serine-Threonine Kinases / chemistry*
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein Subunits
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • RNA, Small Interfering / genetics
  • Sequence Homology, Amino Acid
  • Spindle Apparatus / physiology*

Substances

  • Knl1 protein, human
  • Microtubule-Associated Proteins
  • Nuclear Pore Complex Proteins
  • Protein Subunits
  • Proto-Oncogene Proteins
  • RNA, Small Interfering
  • TPR protein, human
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • BUB1 protein, human
  • Bub1 spindle checkpoint protein
  • Protein Serine-Threonine Kinases