Zacharias M, Luty B A, Davis M E, McCammon J A
University of Houston, Department of Chemistry, TX 77204-5641.
J Mol Biol. 1994 May 6;238(3):455-65. doi: 10.1006/jmbi.1994.1304.
The N-terminal domain of the phage lambda repressor binds as a dimer to its palindromic DNA operator sequence. In addition to a helix-turn-helix DNA recognition motif, the first six amino acids of the phage lambda repressor form a flexible peptide segment which wraps around DNA. Site-directed mutagenesis studies have shown that amino acid replacements or partial removal of the arm structure, or changes in the DNA sequence contacting the N-terminal arm, can lower the repressor-operator binding affinity by several orders of magnitude. The finite-difference Poisson-Boltzmann approach in combination with a conformational search procedure was used to study energetic contributions of the lambda arm to repressor-operator recognition based on the high resolution X-ray structure. It allows for the local relaxation of the structure upon changing the DNA sequence in the lambda arm binding region. A simplified potential energy function including torsional, truncated Lennard-Jones and approximate electrostatic terms is used in the initial step to screen out energetically unfavorable structures. The electrostatic energy of selected conformations is subsequently calculated more accurately using the finite-difference Poisson-Boltzmann approach. The method was applied to study the effect of a C-->T mutation at position 6 of the consensus half-site of the operator. This base-pair contacts Lys4 which is part of the arm segment. Keeping only the Lys4 side-chain mobile and with the wild-type DNA operator sequence, several conformations close to the X-ray structure were identified as those with lowest energy. In the case of the DNA mutation, lowest energy conformations differed significantly from those selected for the wild-type sequence. These initial calculations indicate that the approach might be a useful tool to estimate conformational and energetic effects upon mutagenesis of protein-DNA complexes.
噬菌体λ阻遏物的N端结构域以二聚体形式与其回文DNA操纵序列结合。除了螺旋-转角-螺旋DNA识别基序外，噬菌体λ阻遏物的前六个氨基酸形成一个柔性肽段，该肽段环绕DNA。定点诱变研究表明，氨基酸替换或臂结构的部分去除，或与N端臂接触的DNA序列的改变，可使阻遏物-操纵序列的结合亲和力降低几个数量级。基于高分辨率X射线结构，采用有限差分泊松-玻尔兹曼方法结合构象搜索程序，研究λ臂对阻遏物-操纵序列识别的能量贡献。它允许在改变λ臂结合区域的DNA序列时结构的局部松弛。在初始步骤中使用简化的势能函数，包括扭转、截断的 Lennard-Jones 和近似静电项，以筛选出能量上不利的结构。随后使用有限差分泊松-玻尔兹曼方法更准确地计算所选构象的静电能。该方法用于研究操纵序列共有半位点第6位的C→T突变的影响。这个碱基对与作为臂段一部分的赖氨酸4接触。仅保持赖氨酸4侧链可移动，并使用野生型DNA操纵序列，确定了几个接近X射线结构的构象为能量最低的构象。在DNA突变的情况下，能量最低的构象与为野生型序列选择的构象有显著差异。这些初步计算表明，该方法可能是估计蛋白质-DNA复合物诱变时构象和能量效应的有用工具。
