Meierhan D, el-Ariss C, Neuenschwander M, Sieber M, Stackhouse J F, Allemann R K
Department of Chemistry, ETH-Zurich, Switzerland.
Biochemistry. 1995 Sep 5;34(35):11026-36. doi: 10.1021/bi00035a008.
Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.
尽管MASH-1和MyoD的碱性螺旋-环-螺旋（BHLH）结构域在序列上具有高度相似性，但它们参与不同的生物学过程。为了确定这两种蛋白质在DNA结合特异性上可能存在的差异，我们通过圆二色光谱法和电泳迁移率变动分析（EMSA）研究了MASH-1的DNA结合特性。与DNA结合后，MASH-1的BHLH结构域经历了从主要未折叠状态到主要为α-螺旋形式的构象变化，令人惊讶的是，这种变化与特定的DNA序列无关。添加20%的2,2,2-三氟乙醇也能诱导相同的构象转变。从EMSA的半饱和点确定了全长MASH-1与各种寡核苷酸复合物的表观解离常数（KD）。MASH-1以二聚体形式与含有E-box的DNA序列高亲和力结合（KD = 1.4 - 4.1 x 10(-14) M2）。然而，DNA结合的特异性较低。MASH-1与最高亲和力E-box序列之间复合物的解离常数（KD = 1.4 x 10(-14) M2）仅比与完全不相关的DNA序列（KD = 约1 x 10(-13) M2）小10倍。与普遍存在的E12蛋白形成异二聚体并没有显著提高MASH-1的DNA结合特异性。MASH-1和MyoD表现出相似的结合位点偏好，这表明它们不同的靶基因特异性不能仅通过不同的DNA结合来解释。基于MyoD的BHLH结构域的已知晶体结构，对这些发现提供了一种解释。
