Understanding the Role of KH Domain in Helicases

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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.