Preferential Heterodimeric Parallel Coiled-coil Formation by Synthetic Max and c-Myc Leucine Zippers: A Description of Putative Electrostatic Interactions Responsible for the Specificity of Heterodimerization

Abstract

The oncoprotein c-Myc must heterodimerize with Max to bind DNA and perform its oncogenic activity. The c-Myc – Max heterodimer binds DNA through a basic helix-loop-helix leucine zipper (b-HLH-zip) motif and it is proposed that leucine zipper domains could, in concert with the HLH regions, provide the specificity and stability of the b-HLH-zip motif. In this context, we have synthesized the peptides corresponding to the leucine zipper domains of Max and c-Myc with a N-terminal Cys-Gly-Gly linker and studied their dimerization behavior using reversed-phase HPLC and CD spectroscopy. The preferential formation of a fully helical parallel c-Myc – Max heterodimeric coiled-coil was observed under air-oxidation and redox conditions at neutral pH. We show that the stability and the helicity of the disulfide-linked c-Myc – Max heterostranded coiled-coil is modulated by pH, with a maximum around pH 4.5, supporting the existence of stabilizing and specific interhelical electrostatic interactions. We present a molecular model of the c-Myc – Max heterostranded coiled-coil describing potential electrostatic interactions responsible for the specificity of the interaction, the main feature being putative buried electrostatic interactions between a histidine side-chain (in the Max leucine zipper) and two glutamic acid side-chains (in the c-Myc leucine zipper) at the heterodimer interface. This model is supported by the fact that the apparent p Ka (as determined by [ 1H]-NMR spectroscopy) of this histidine side-chain at 25°C is 0.42 (±0.05) p Ka units higher in the folded form than in the unfolded form. This indicates that the charged histidine side-chain contributes approximately 0.57 (±0.07) kcal/mol (2.38 (±0.30) kJ/mol) of stabilization free energy to the c-Myc – Max heterostranded coiled-coil through favorable electrostatic interaction.