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.

The protein kinase PKR is a key factor in innate immunity.1 In the presence of long stretches of A-form dsRNA, PKR is activated to undergo autophosphorylation.2,3 Once activated, PKR phosphorylates translation initiation factor eIF-2α, which blocks translation and thus pathogen replication. PKR is 551 amino acids and comprised of an N-terminal dsRNA-binding domain (dsRBD), which has two tandem copies of the dsRNA-binding motif (dsRBM), and a C-terminal kinase domain. The dsRBM is a non-sequence-specific 65-70 amino acid RNA binding motif that has an αβββα secondary structure.4-6 It binds non-sequence specifically to A-form dsRNA primarily in its wide and shallow minor groove.7-9 These interactions involve hydrogen bonds with the 2′OH groups, which leads to discrimination against RNA-DNA hybrids.7 The dsRBM is present in many important proteins. For instance, Dicer and Drosha, which process highly structured miRNA precursors, have single copies of the dsRBM, as does E. coli RNase III.4-6 In addition, many proteins have multiple dsRBM copies, which are typically present in tandem: for example, PKR has two tandem copies of the dsRBM; the RNA-editing human adenosine deaminases (ADAR1 and ADAR2) have three and two copies, respectively; and the developmental protein Staufen has five tandem copies, with dsRBM3 and dsRBM4 being involved in dsRNA binding.10-13 While the dsRBM binds strongly to perfect dsRNA and also with varying degrees of affinity to dsRNAs with imperfections, it is not clear which RNAs activate PKR. This is an important issue as most cellular and viral RNA are not perfectly double-stranded; for instance, pri- and pre-miRNAs contain helical defects,14 as do most PKR regulators.15 Studies from our lab and others support PKR binding to and being activated by RNA structures with bulges, hairpin loops, and pseudoknots, and by weakly structured RNAs with a 5′-triphosphate.16-19 In at least several cases, the RNAs have complex folds that mimic A-form dsRNA.16,19,20 In addition, ITC studies have been conducted on PKR binding to the viral RNA inhibitors VAI RNA and EBER RNA, which have complex secondary and tertiary structures, and have revealed tight and specific binding.21-23 We recently studied interaction of PKR with model RNAs containing bulges and internal loops.24 Bulges invariably decreased PKR activation, although trans-bulges, in which flanking helices are on opposite sides of a central helix, inhibited activation more than cis-bulges, in which flanking helices are on the same side of a central helix. These studies extended earlier EMSA experiments, which had suggested that the dsRBM interacts with a bulged RNA in a weak fashion.25 In the present study, we conduct thermodynamic and structural investigations of the binding of PKR and its N-terminal dsRBD, termed ‘p20’, to various chimeric duplexes using EMSA, ITC, and SAXS. The chimeric duplexes contain a central, minimally sized dsRNA binding site flanked by RNA-DNA hybrid “arms”. In addition, certain central segments are dsRNA with either A3- or A6-bulges, while others are purely RNA-DNA hybrids. We show that only the perfect dsRNA-containing chimeric duplex is capable of binding the dsRBD from PKR tightly, while all other sites bind weakly. We further find that p20 straightens bulged RNAs to geometry consistent with A-form. These findings suggest ways in which PKR may interact with biological RNAs having complex secondary and tertiary structures.