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.
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 pKa (as determined by [1H]-NMR spectroscopy) of this histidine side-chain at 25 degrees C is 0.42 (+/- 0.05) pKa 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.
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003198:Computer Simulation; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D018257:Helix-Loop-Helix Motifs; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D016350:Leucine Zippers; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D004220:Disulfides; C067850:Myc associated factor X; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D014157:Transcription Factors