Structure and Function of KH Domain in DDX43 and DDX53 Cancer Antigen Helicases
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DDX43 and DDX53 are two cancer/testis antigens that are overexpressed in various tumors, but absent or expressed at very low levels in normal tissues; therefore, both are proposed to be valid candidates for therapeutic targets. DDX43 and DDX53 belong to the DEAD-box RNA helicase family, and contain a K-homology (KH) domain in their N-terminal regions and a helicase core domain in their C-terminal regions. The KH domain was first characterized in the human heterogeneous nuclear ribonucleoprotein K (hnRNP K). The typical function of KH domain is to bind single-stranded (ss) RNA or ssDNA in a sequence specific manner. The helicase core domain contains two conserved RecA-like domains and unwinds double-strand nucleic acids in a sequence-unspecific manner; therefore, we hypothesize that the KH domain may play a critical role in sequence-specific function of DDX43 and DDX53. In this study, we expressed and purified DDX43-KH and DDX53-KH domains using Ni-NTA column chromatography and size exclusion chromatography. Using electrophoretic mobility shift assay (EMSA), we found that DDX43-KH domain binds ssDNA and ssRNA substrates efficiently but not blunt-end double-stranded (ds) DNA or dsRNA substrates. We applied 1H-15N HSQC NMR spectroscopy to determine the binding affinity of DDX43-KH with dT5, dA5, dC5, dG5 and rU5 oligonucleotides, and found that there are significant chemical shift changes in the HSQC spectra for pyrimidines (dT5, dC5, rU5), but not for purines (dA5 and dG5). Titrations with dT10 and dT5 oligonucleotides revealed that the GRGG loop in DDX43-KH domain is involved in protein-nucleic acids interactions. In addition, we found that the Ala81, adjacent to the GRGG loop, is involved in the binding. A81G and A81S mutations reduced protein stability and binding affinity. To further identify the specific nucleotides bound by the DDX43-KH and DDX53-KH domains, we used SELEX (Systematic evolution of ligands by exponential enrichment) and ChIP-seq (Chromatin Immunoprecipitation Sequencing)/CLIP-seq (Cross-linking immunoprecipitation-sequencing), and found CT-rich sequences are commonly occurring motifs for DDX43-KH domain. However, GTTGA, TG/TTG/TG/T motifs are also common in the SELEX and CLIP samples. Finally, NMR spectroscopy showed that GTTGT has the lowest Kd value (0.0435 mM); suggesting, DDX43-KH binds to TG-rich sequences with high affinity and might partially dictate the substrate specificity for the full length DDX43 protein.
DegreeMaster of Science (M.Sc.)
SupervisorWu, Yuliang; Cygler, Miroslaw
CommitteeLee, Jeremy; Dmitriev, Oleg; Sanders, David
Copyright DateApril 2020
KH domain, DDX43, DDX53, substrate specificity, EMSA, SELEX, ChIP-Seq, CLIP-Seq, NMR spectroscopy