Understanding the Role of KH Domain in Helicases

我的研究计划的长期目标是解旋酶的结构和功能表征。本提案的短期目标是了解KH结构域在解旋酶中的作用。背景：K同源(KH)结构域20年前首次在人类异质性核糖核蛋白K(HnRNP K)中被发现，随后在多种蛋白质中发现。KH结构域长约70个氨基酸，中间有一个与核酸接触的保守共有序列GXXG。解旋酶是解开和重塑结构DNA或RNA的分子马达。DEAD-box解旋酶DDX43和DDX53是仅有的两个含有KH结构域的解旋酶。最新进展：我们发现KH结构域是DDX43的S功能所必需的(JBC，2017)。我们还纯化了重组的DDX53蛋白，发现1)DDX53蛋白以单体和四聚体形式存在，而DDX43只以单体形式存在；2)DDX53与DDX43一致，可以解开3‘末端而不是5’末端双链DNA；目的1.确定KH结构域在DDX53解旋酶中的作用。DDX53与DDX43既相似又不同，这促使我们问他们的KH结构域是否造成了差异。我们将1)纯化DDX53‘-S KH结构域(50-122aa)并检测其寡聚状态和核酸结合情况；2)纯化DDX53’S解旋酶核心区(191aa-631aa)并研究其是否具有解旋酶功能；3)改变GYSG环中保守的甘氨酸并研究DDX53‘S的寡聚状态和解离活性；目的2.确定KH结构域结合的核酸。DDX43有GRGG环，DDX53有GYSG环，我们推测它们的KH结构域可能提供了核酸底物特异性。为了鉴定KH结构域结合的核酸，将使用两种方法：体外指数富集型配体系统进化(SELEX)和体内交联-免疫沉淀纯化结合KH结构域的DNA/RNA，然后进行高通量测序。目的3.确定KH结构域和解旋酶与核酸结合的结构。我们观察到了DDX43KH结构域蛋白的针状晶体。我们现在正在优化条件，以获得更大的晶体。我们还将克隆DDX53 KH结构域，并纯化其蛋白质用于晶体筛选，以及两个全长解旋酶。一旦获得合适的晶体，蛋白质/核酸结构将通过位于南加州大学校园的加拿大光源的X射线结晶学来确定。意义：虽然KH结构域已在多种蛋白质中被发现，但在解旋酶中尚未见报道。因此，我们对这些解旋酶中KH结构域的结构和功能的研究不仅将有助于揭示KH结构域与核酸的特异性相互作用，而且还将为了解解旋酶的机制提供帮助。

The long-term goal of my research program is structural and functional characterization of helicases. The short-term objective of the current proposal is to understand the role of the KH domain in helicases. Background: The K Homology (KH) domain was first identified in the human heterogeneous nuclear ribonucleoprotein K (hnRNP K) two decades ago, and has subsequently been found in a variety of proteins. The KH domain is approximately 70 amino acids long with a conserved consensus sequence GXXG in the middle that contacts nucleic acids. Helicases are molecular motors that unwind and remodel structured DNA or RNA. DEAD-box helicases DDX43 and DDX53 are the only two helicases known to contain the KH domain. Recent progress: We found that the KH domain is essential for DDX43's functions (JBC, 2017). We also purified recombinant DDX53 protein and found 1) DDX53 protein exists in monomeric and tetrameric forms, whereas DDX43 is monomeric only, 2) DDX53 could unwind 3' tailed but not 5' tailed duplex DNA, consistent with DDX43, and 3) DDX53 failed to unwind RNA duplex substrates while DDX43 could. Objective 1. Determine the role of the KH domain in DDX53 helicase. DDX53 is both similar to and different from DDX43, which led us to ask whether their KH domains make the difference. We will 1) purify DDX53's KH domain (50-122 aa) and examine its oligomeric status and nucleic acid binding, 2) purify DDX53's helicase core domain (191-631 aa) and study whether it can function as a helicase, 3) change the conserved glycine in the GYSG loop and investigate DDX53's oligomeric status and unwinding activity, and 4) switch the KH domains of DDX43 and DDX53. Objective 2. Determine the nucleic acid bound by the KH domains. DDX43 has a GRGG loop and DDX53 has a GYSG loop, and we hypothesize that their KH domains may provide nucleic acid substrate specificity. To identify the nucleic acid bound by the KH domains, two approaches will be used: in vitro Systematic Evolution of Ligands by EXponential enrichment (SELEX) and in vivo crosslinking-immunoprecipitation to purify DNA/RNAs bound to the KH domain followed by high-throughput sequencing. Objective 3. Determine the structure of the KH domain and helicase with the nucleic acid bound. We have observed needle-like crystals for DDX43 KH domain protein. We are now optimizing conditions to obtain bigger crystals. We will also clone the DDX53 KH domain and purify its protein for crystal screening, as well as the two full-length helicases. Once suitable crystals are obtained, the protein/nucleic acid structure will be determined by X-ray crystallography at the Canadian Light Source on the UofS campus. Significance: Although the KH domain has been found in a variety of proteins, it has not been reported in helicases. Thus, our structural and functional studies of the KH domains in these helicases will not only shed light on KH domain-nucleic acid specific interactions, but also provide mechanistic insights of helicases.