Specificity of the Double-Stranded RNA-Binding Domain from the RNA-Activated Protein Kinase PKR for Double-Stranded RNA: Insights from Thermodynamics and Small-Angle X-ray Scattering

详细信息    查看全文

• 作者：Sunita Patel ; Joshua M. Blose ; Joshua E. Sokoloski ; Lois Pollack ; Philip C. Bevilacqua
• 刊名：Biochemistry
• 出版年：2012
• 出版时间：November 20, 2012
• 年：2012
• 卷：51
• 期：46
• 页码：9312-9322
• 全文大小：470K
• 年卷期：v.51,no.46(November 20, 2012)
• ISSN：1520-4995

文摘

The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed 鈥減20鈥?for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA鈥揇NA hybrid segments and a central dsRNA segment, which was either unbulged (鈥減erfect鈥? or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K_d 30 nM) and weaker binding to the bulged duplex (K_d 2鈥? 渭M). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD鈥檚 ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.