High affinity dimerization by Ski involves parallel pairing of a novel bipartite alpha-helical domain

J Biol Chem. 1997 Dec 12;272(50):31855-64. doi: 10.1074/jbc.272.50.31855.

Abstract

c-Ski protein possesses a C-terminal dimerization domain that was deleted during the generation of v-ski, and has been implicated in the increased potency of c-ski in cellular transformation compared with the viral gene. The domain is predicted to consist of an extended alpha-helical segment made up of two motifs: a tandem repeat (TR) consisting of five imperfect repeats of 25 residues each and a leucine zipper (LZ) consisting of six heptad repeats. We have examined the structure and dimerization of TR or LZ individually or the entire TR-LZ domain. Using a quenched chemical cross-linking method, we show that the TR dimerizes with moderate efficiency (Kd = 4 x 10(-6) M), whereas LZ dimerizes poorly (Kd > 2 x 10(-5) M). However, the entire TR-LZ domain dimerizes efficiently (Kd = 2 x 10(-8) M), showing a cooperative effect of the two motifs. CD analyses indicate that all three proteins contain predominantly alpha-helices. Limited proteolysis of the TR-LZ dimer indicates that the two helical motifs are linked by a small loop. Interchain disulfide bond formation indicates that both the LZ and TR helices are oriented in parallel. We propose a model for the dimer interface in the TR region consisting of discontinuous clusters of hydrophobic residues forming "leucine buttons."

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Chickens
  • Circular Dichroism
  • DNA-Binding Proteins / chemistry*
  • Dimerization
  • Molecular Sequence Data
  • Protein Conformation
  • Protein Structure, Secondary
  • Proto-Oncogene Proteins / chemistry*

Substances

  • DNA-Binding Proteins
  • Proto-Oncogene Proteins