Gross P, Yee A A, Arrowsmith C H, Macgregor R B
Department of Pharmaceutical Sciences, University of Toronto, Ontario, Canada.
Biochemistry. 1998 Jul 7;37(27):9802-11. doi: 10.1021/bi9731448.
Quantitative hydroxyl radical footprinting and fluorescence polarization measurements have been used to determine the dissociation constants (Kd) of complexes between the ets domain of the murine transcription factor PU.1 and three different DNA fragments. Two natural PU.1 binding sites, the SV40 enhancer site and the lambdaB motif of Iglambda2-4 enhancer, were used as well as the PU.1 binding site present in the crystallized PU.1-DNA complex. With the use of quantitative hydroxyl radical footprinting we obtained binding isotherms for individual protected nucleotides and contact sites on both strands of the DNA. Kd values of (1.53 +/- 0. 12) x 10(-)8 M were found for the lambdaB element, (3.60 +/- 0.65) x 10(-)8 M for the SV40 enhancer site, and (2.28 +/- 0.27) x 10(-)8 M for the sequence used in the crystal structure. In addition, the binding of a second protein, the DNA binding domain of IRF4, to the lambdaB site by itself and in the presence of PU.1 was analyzed. The IRF4 DBD shows three footprints on the TTCC strand and one footprint on the GGAA strand of the lambdaB element. The dissociation constant for the binary IRF4 DBD-lambdaB complex equals (5.59 +/- 0.60) x 10(-)7 M. The Kd value of the IRF4-lambdaB interaction is reduced by a factor of 5 in the presence of two different DNA-bound PU.1 protein constructs, PU.1 DBD and a PU.1 construct containing the PEST domain (PU.1-PEST). A similar decrease of the Kd value was observed for the binding of PU.1-PEST in the presence of DNA-bound IRF4 DBD demonstrating a cooperative interaction between the PU. 1-PEST and IRF4 DBD. On the basis of the hydroxyl radical footprints in the ternary PU.1/IRF4/lambdaB complex, a model for the interactions between the two proteins and the lambdaB site was developed. The DNA binding domains of both proteins bind the DNA in the major groove with potential protein-protein interactions near the intervening minor groove.
定量羟基自由基足迹法和荧光偏振测量已被用于确定小鼠转录因子PU.1的ets结构域与三个不同DNA片段之间复合物的解离常数（Kd）。使用了两个天然的PU.1结合位点，即SV40增强子位点和Iglambda2 - 4增强子的lambdaB基序，以及存在于结晶的PU.1 - DNA复合物中的PU.1结合位点。通过定量羟基自由基足迹法，我们获得了DNA两条链上各个受保护核苷酸和接触位点的结合等温线。发现lambdaB元件的Kd值为(1.53±0.12)×10⁻⁸ M，SV40增强子位点的Kd值为(3.60±0.65)×10⁻⁸ M，晶体结构中使用的序列的Kd值为(2.28±0.27)×10⁻⁸ M。此外，还分析了第二种蛋白质，即IRF4的DNA结合结构域，自身以及在存在PU.1的情况下与lambdaB位点的结合。IRF4 DBD在lambdaB元件的TTCC链上显示出三个足迹，在GGAA链上显示出一个足迹。二元IRF4 DBD - lambdaB复合物的解离常数等于(5.59±0.60)×10⁻⁷ M。在存在两种不同的与DNA结合的PU.1蛋白构建体，即PU.1 DBD和含有PEST结构域的PU.1构建体（PU.1 - PEST）的情况下，IRF4与lambdaB相互作用的Kd值降低了5倍。在存在与DNA结合的IRF4 DBD的情况下，观察到PU.1 - PEST结合的Kd值也有类似降低，这表明PU.1 - PEST与IRF4 DBD之间存在协同相互作用。基于三元PU.1/IRF4/lambdaB复合物中的羟基自由基足迹，建立了两种蛋白质与lambdaB位点之间相互作用的模型。两种蛋白质的DNA结合结构域都在大沟中结合DNA，在中间小沟附近存在潜在的蛋白质 - 蛋白质相互作用。
