Analysis of high-affinity binding of protein kinase R to double-stranded RNA.
Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to double-stranded RNA (dsRNA). Our previous analysis of binding of PKR to dsRNAs ranging from 20 to 40 bp supports a dimerization model for activation in which 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high-affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt concentrations but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode in which both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, United States.
Bushra
B
Husain
Ishita
I
Mukerji
James L
JL
Cole
D004789:Enzyme Activation; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D012330:RNA, Double-Stranded / Q000378:metabolism*; D014461:Ultracentrifugation; D019892:eIF-2 Kinase / Q000737:chemistry / Q000378:metabolism*
D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase