Defining the eukaryotic cytosolic chaperonin-binding sites in human tubulins

J Mol Biol. 2000 Nov 17;304(1):81-98. doi: 10.1006/jmbi.2000.4177.

Abstract

The actins and tubulins are the obligate substrates in vivo of the chaperonin-containing TCP-1 (CCT). The precise elements of recognition between the chaperonin and its substrates remain largely unknown. We have used a solid phase peptide binding assay to screen the human alpha, beta and gamma-tubulin sequences for CCT recognition. Multiple regions seem to be implicated in interactions between tubulins and CCT. These potential CCT-binding sites are highly dispersed throughout the primary sequences of the human tubulins. In addition, using site-directed mutagenesis we assessed the contribution of the selected residues in the C-terminal domain of beta-tubulin to CCT binding. Various hot spots have been identified even though, in each case, their replacement by alanine does not reduce dramatically the total affinity of beta-tubulin for CCT. The CCT-binding information in the tubulins is probably confined to multiple specific regions each having weak or moderate affinity for CCT apical domains. The main binding region seems to be located between residues 263 and 384, but there are no single amino acid residues in this region, which make large contributions to the binding energy, although we have detected a minor contribution by F377. These biochemical results are understandable in the context of our recent structural analysis of CCT-tubulin complexes by cryo-electron microscopy and image reconstruction, which shows that, in one stage of an in vitro binding reaction between apo-CCT and tubulin diluted from guanidinium chloride, ten major, stable contacts between tubulin and CCT are involved. Therefore, specificity is achieved through the co-operation of many specific, albeit weak, interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Chaperonins / classification
  • Chaperonins / metabolism*
  • Cytosol / chemistry
  • Cytosol / metabolism*
  • DNA, Complementary / genetics
  • Humans
  • Male
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation / genetics
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity
  • Testis / cytology
  • Thermodynamics
  • Tubulin / chemistry*
  • Tubulin / genetics
  • Tubulin / metabolism*

Substances

  • DNA, Complementary
  • Peptide Fragments
  • Recombinant Fusion Proteins
  • Tubulin
  • Chaperonins