Lavigne P, Kondejewski L H, Houston M E, Sönnichsen F D, Lix B, Skyes B D, Hodges R S, Kay C M
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Mol Biol. 1995 Dec 1;254(3):505-20. doi: 10.1006/jmbi.1995.0634.
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.
癌蛋白c-Myc必须与Max形成异源二聚体才能结合DNA并发挥其致癌活性。c-Myc-Max异源二聚体通过一个碱性螺旋-环-螺旋亮氨酸拉链（b-HLH-zip）基序结合DNA，有人提出亮氨酸拉链结构域可以与HLH区域协同作用，为b-HLH-zip基序提供特异性和稳定性。在此背景下，我们合成了与Max和c-Myc的亮氨酸拉链结构域相对应的肽段，并在其N端连接了Cys-Gly-Gly接头，然后使用反相高效液相色谱和圆二色光谱研究了它们的二聚化行为。在空气氧化和中性pH的氧化还原条件下，观察到了完全螺旋的平行c-Myc-Max异源二聚体卷曲螺旋的优先形成。我们发现，二硫键连接的c-Myc-Max异源双链卷曲螺旋的稳定性和螺旋度受pH调节，在pH 4.5左右达到最大值，这支持了稳定且特异的螺旋间静电相互作用的存在。我们提出了一个c-Myc-Max异源双链卷曲螺旋的分子模型，该模型描述了负责相互作用特异性的潜在静电相互作用，其主要特征是在异源二聚体界面处，一个组氨酸侧链（在Max亮氨酸拉链中）与两个谷氨酸侧链（在c-Myc亮氨酸拉链中）之间存在假定的埋藏静电相互作用。该模型得到了以下事实的支持：在25℃下，该组氨酸侧链在折叠形式下的表观pKa（通过[1H]-核磁共振光谱测定）比未折叠形式高0.42（±0.05）个pKa单位。这表明，带电荷的组氨酸侧链通过有利的静电相互作用为c-Myc-Max异源双链卷曲螺旋贡献了约0.57（±0.07）kcal/mol（2.38（±0.30）kJ/mol）的稳定自由能。
