Binding Polymorphism in the DNA Bound State of the Pdx1 Homeodomain

The subtle effects of DNA-protein recognition are illustrated in the homeodomain fold. This is one of several small DNA binding motifs that, in spite of limited DNA binding specificity, adopts crucial, specific roles when incorporated in a transcription factor. The homeodomain is composed of a 3-helix domain and a mobile N-terminal arm. Helix 3 (the recognition helix) interacts with the DNA bases through the major groove, while the N-terminal arm becomes ordered upon binding a specific sequence through the minor groove. Although many structural studies have characterized the DNA binding properties of homeodomains, the factors behind the binding specificity are still difficult to elucidate. A crystal structure of the Pdx1 homeodomain bound to DNA (PDB 2H1K) obtained previously in our lab shows two complexes with differences in the conformation of the N-terminal arm, major groove contacts, and backbone contacts, raising new questions about the DNA recognition process by homeodomains. Here, we carry out fully atomistic Molecular Dynamics simulations both in crystal and aqueous environments in order to elucidate the nature of the difference in binding contacts. The crystal simulations reproduce the X-ray experimental structures well. In the absence of crystal packing constraints, the differences between the two complexes increase during the solution simulations. Thus, the conformational differences are not an artifact of crystal packing. In solution, the homeodomain with a disordered N-terminal arm repositions to a partially specific orientation. Both the crystal and aqueous simulations support the existence of different stable binding conformers identified in the original crystallographic data with different degrees of specificity. We propose that protein-protein and protein-DNA interactions favor a subset of the possible conformations. This flexibility in DNA binding may facilitate multiple functions for the same transcription factor.

DNA-蛋白质识别的细微效应可通过同源结构域（homeodomain）折叠加以阐释。同源结构域是一类小型DNA结合基序之一，尽管其DNA结合特异性有限，但当整合入转录因子（transcription factor）时，会发挥关键且特定的功能。同源结构域由三螺旋结构域与可移动的N端臂组成。螺旋3（即识别螺旋）通过DNA大沟与DNA碱基相互作用，而N端臂在通过小沟结合特定序列时会变得有序。尽管已有诸多结构研究对同源结构域的DNA结合特性进行了表征，但结合特异性背后的机制仍难以阐明。本实验室此前获得的结合DNA的Pdx1同源结构域晶体结构（PDB 2H1K）显示，两个复合物在N端臂构象、大沟接触以及主链接触方面存在差异，这为同源结构域的DNA识别过程提出了新的疑问。在此，我们分别在晶体与水溶液环境中开展全原子分子动力学（Molecular Dynamics）模拟，以阐明结合接触差异的本质。晶体模拟很好地复现了X射线实验结构。在无晶体堆积限制的情况下，两个复合物之间的差异在溶液模拟过程中有所增大。因此，这些构象差异并非晶体堆积所产生的人工假象。在溶液中，N端臂无序的同源结构域会重新定位至部分特异性的取向。晶体与水溶液模拟均支持原始晶体学数据中鉴定出的、具有不同特异性程度的多种稳定结合构象异构体的存在。我们提出，蛋白质-蛋白质与蛋白质-DNA相互作用会倾向于选择部分可能的构象。这种DNA结合的灵活性或许有助于同一转录因子实现多种功能。