Molecular organization of the cullin E3 ligase adaptor KCTD11

Biochimie. 2011 Apr;93(4):715-24. doi: 10.1016/j.biochi.2010.12.014. Epub 2011 Jan 13.

Abstract

The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cell Cycle Proteins
  • Cullin Proteins / chemistry
  • Cullin Proteins / genetics
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed / methods
  • Potassium Channels / chemistry*
  • Potassium Channels / genetics*
  • Protein Binding / genetics
  • Protein Isoforms / chemistry
  • Protein Isoforms / genetics
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Transferases
  • Ubiquitin / chemistry
  • Ubiquitin-Protein Ligases / chemistry*

Substances

  • Cell Cycle Proteins
  • Cullin Proteins
  • Potassium Channels
  • Protein Isoforms
  • Recombinant Proteins
  • Ubiquitin
  • KCTD11 protein, human
  • Transferases
  • Ubiquitin-Protein Ligases