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

Area of Science:

• Protein structure and dimerization
• Molecular biology
• Biochemistry

Background:

• The C-terminal dimerization domain of c-Ski is crucial for its cellular transformation potency.
• This domain contains tandem repeat (TR) and leucine zipper (LZ) motifs, which are alpha-helical.
• Understanding the dimerization of these motifs is key to c-Ski's function.

Purpose of the Study:

• To investigate the structure and dimerization properties of the TR and LZ motifs individually and combined.
• To elucidate the cooperative effects between the TR and LZ domains in c-Ski.
• To propose a structural model for the c-Ski dimerization interface.

Main Methods:

• Quenched chemical cross-linking to determine dimerization efficiency (Kd).
• Circular dichroism (CD) spectroscopy to analyze secondary structure.
• Limited proteolysis to study domain linkage.
• Interchain disulfide bond formation to assess helix orientation.

Main Results:

• TR dimerizes with moderate efficiency (Kd = 4 x 10(-6) M); LZ dimerizes poorly (Kd > 2 x 10(-5) M).
• The combined TR-LZ domain exhibits efficient dimerization (Kd = 2 x 10(-8) M) due to cooperative effects.
• CD analysis confirms predominantly alpha-helical structures for TR, LZ, and TR-LZ.
• Proteolysis and disulfide bond formation indicate parallel orientation of TR and LZ helices, linked by a loop.

Conclusions:

• The c-Ski dimerization domain functions cooperatively, with TR and LZ motifs enhancing overall dimerization efficiency.
• A structural model involving "leucine buttons" in the TR region is proposed for the dimer interface.
• These findings provide insights into the molecular mechanisms underlying c-Ski's role in cellular transformation.