Protein Dynamics in Homeodomain/DNA Complexes

描述(由申请人提供)：基本的细胞活动，如基因的转录、复制、重组和修复，需要DNA和DNA结合蛋白的非共价相互作用。控制蛋白质-DNA相互作用的潜在分子识别过程是复杂的，尚未完全了解。特别是，在我们对分子动力学在蛋白质/DNA相互作用中扮演的角色的认识和理解方面存在着关键和实质性的差距。分子识别领域的一个普遍问题是，结构研究揭示的关于络合物形成自由能的熵分量相对较少。因此，通过开展研究来补充可用的结构信息是非常重要的，这些研究旨在阐明关于蛋白质/DNA界面构象动力学的性质和贡献的细节。我们建议使用溶液状态核磁共振波谱来表征蛋白质/DNA复合体模型体系中氨基酸侧链的动力学。被选择用于研究的模型系统是所谓的K50类同源结构域蛋白，它的定义是那些在DNA识别螺旋的50位有赖氨酸残基的同源结构域。同源结构域基序是描述蛋白质/DNA相互作用的一个非常重要的模型系统，功能和结构研究都提供了大量关于同源结构域/DNA相互作用的信息。这些结构研究表明了一种保守的全球折叠和对接排列，但关于蛋白质-DNA界面上关键氨基酸残基的作用以及侧链运动的程度和意义仍然存在基本问题。这项研究的一般假设是，蛋白质侧链动力学的调节(而不是固定)在(1)在DNA和同源DNA结合蛋白之间建立互补接口，以及(2)允许给定的DNA结合蛋白识别共识和非共识DNA序列中发挥重要作用。该项目的具体目标是：(1)确定果蝇双核蛋白与含有共识结合位点TAATCC的DNA双链结合的同源结构域的侧链动力学；(2)确定与非共识DNA位点结合的双核同源结构域的侧链动力学；以及(3)确定Pitx2同源域/DNA复合体的结构和动力学。这些特定的目标中的每一个都需要确定同源结构域/DNA复合体的结构；到目前为止，还没有K50类同源结构的报道。显然，同源域模型不能完全代表所有的蛋白质/DNA相互作用。然而，本文提出的蛋白质动力学的全面研究将有助于建立一个关于蛋白质运动的性质及其在蛋白质/DNA识别中的意义的知识基础，未来的工作可以在此基础上进行。

DESCRIPTION (provided by applicant): Fundamental cellular activities such as the transcription, replication, recombination and repair of genes require the non-covalent interaction of DNA and DNA-binding proteins. The underlying molecular recognition processes governing protein-DNA interactions are complex and not yet fully understood. In particular, critical and substantial gaps exist in our knowledge and understanding of the role played by molecular dynamics in protein/DNA interactions. A general problem in the field of molecular recognition is that structural studies reveal relatively little about the entropic component of the free energy of complex formation. Thus, it is very important to complement available structural information by undertaking studies designed to elucidate details concerning the nature and contributions of conformational dynamics in the protein/DNA interface. We propose to use solution-state NMR spectroscopy to characterize the dynamics of amino acid side chains in model systems of protein/DNA complexes. The model system chosen for study is the so-called K50 class of homeodomain proteins, which is defined as those homeodomains that have a lysine residue at position 50 in the DNA recognition helix. The homeodomain motif has been a very important model system for characterizing protein/DNA interactions, and a wealth of information concerning homeodomain/DNA interactions has been provided by both functional and structural studies. These structural studies have demonstrated a conserved global fold and docking arrangement, but fundamental questions remain concerning the roles of key amino acid residues at the protein-DNA interface and about the extent and significance of side chain motions. The general hypotheses of the proposed research are that modulation (as opposed to immobilization) of protein side chain dynamics plays important roles in (1) establishing a complementary interface between DNA and a cognate DNA-binding protein, and (2) allowing a given DNA-binding protein to recognize consensus and non-consensus DNA sequences. The specific aims of the project are: (1) to determine the side chain dynamics of the homeodomain from the Drosophila Bicoid protein, bound to a DNA duplex containing the consensus binding site TAATCC; (2) to determine the side chain dynamics of the Bicoid homeodomain bound to a non-consensus DNA site; and (3) to determine the structure and dynamics of the Pitx2 homeodomain/DNA complex. Each of these specific aims will require a structure determination of the homeodomain/DNA complex; to date no K50-class homeodomain structure has been reported. Clearly, the homeodomain model cannot be fully representative of all protein/DNA interactions. However, the comprehensive studies of protein dynamics that are proposed herein will help to create a foundation of knowledge upon which future work can build, regarding the nature of protein motions and their significance in protein/DNA recognition.