Analysis of high-affinity binding of protein kinase R to double-stranded RNA.

Protein kinase R (PKR) is an important component of the interferon-induced innate immune pathway (1). PKR is synthesized in the cell in a latent, unphosphorylated form and becomes activated and undergoes autophosphorylation upon binding to certain host or pathogen associated RNAs or upon interacting with the protein PACT. Activated PKR phosphorylates eukaryotic Initiation Factor 2α (eIF2α), causing an inhibition of protein synthesis. Many viruses have developed mechanisms to inhibit PKR (2). PKR contains an N-terminal dsRNA binding domain and a C-terminal kinase domain, separated by a flexible linker region. The N-terminal dsRNA binding domain (dsRBD) corresponds to the regulatory domain which consists of two tandem copies of the dsRNA binding motif, dsRBM1 and dsRBM2. This motif binds duplex RNA in a non-sequence specific manner but discriminates against ssRNA and dsDNA (3). Each motif adopts the conserved αβββα fold (4) and they are separated by an unstructured linker of ~20 amino acids. The crystal structure of a dsRNA binding domain complexed with dsRNA reveals a site size of 16 bp (5). The C-terminal kinase domain is the catalytic center. The crystal structure of the PKR kinase domain in complex with eIF2α reveals that the catalytic center has the canonical protein kinase fold. The complex forms a dimer where the interface is in the N-terminal lobe of the kinase domain (6). PKR is regulated by a variety of cellular and viral RNAs that contain duplex regions (7). The most well characterized activators of PKR are simple dsRNAs. There is strong evidence that dimerization plays a crucial role in the mechanism of PKR activation by dsRNA (8, 9). Latent PKR dimerizes weakly in solution but this reaction is sufficient to activate PKR in the absence of RNA (10). An allosteric pathway has been identified that couples the dimer interface with the kinase active site and dimer interface mutations can modulate PKR activation (6, 11). A hallmark of PKR activation by dsRNA is the “bell-shaped” curve where low RNA concentrations activate but higher concentrations are inhibitory (12, 13). These observations support a model in which multiple PKRs assemble on a single dsRNA and high dsRNA concentrations dilute PKR monomers onto separate molecules of dsRNA (14). In the context of regular duplex RNA, a minimum of 30-33 bp of dsRNA are required to activate PKR autophosphorylation and the maximal level of activation increases with duplex length (13, 15). The mechanism of assembly of PKR on duplex RNAs has been extensively investigated. PKR can bind to duplexes as short as 16-18 bp (16, 17). As expected for a non-sequence specific interaction, the binding stoichiometry increases with dsRNA length (13, 15-18). The binding stoichiometries for the dsRBM1 and for a construct containing dsRBM1 and dsRBM2 are greater than predicted from the site size (17, 19). Based on these results, we proposed an overlapping ligand binding model where the motif binds to multiple faces of the dsRNA duplex and overlaps along the helical axis. The increase in binding affinity with increasing dsRNA length can be understood as a statistical consequence of the greater number of configurations available on the longer lattice. The intrinsic binding affinities of a dsRBM1-dsRBM2 construct and full length PKR for a 20 bp RNA are similar (15, 17) but the stoichiometries are lower for the latter. A minimum of 30 bp of dsRNA are required to bind two full length PKR monomers with high affinity, which correlates with the minimal dsRNA capable of activating through autophosphorylation. Binding affinities increase dramatically upon reducing the salt concentration from 200 to 75 mM NaCl and a second PKR can bind to 20 bp duplex at lower salt (15). The non-sequence specific interactions of PKR with RNAs are labile and it is important to use solution biophysical methods to accurately measure the binding parameters at thermodynamic equilibrium. Our previous analyses of PKR binding to dsRNA by sedimentation velocity analytical ultracentrifugation (10, 15) were limited by formation of nonspecific aggregates, even at the minimal RNA concentrations required for detection using absorption optics. Aggregation is enhanced for longer RNAs and at lower salt concentrations. In addition, it is difficult to quantify high affinity binding constants due to the limited sensitivity of the absorbance optics. Here, we have used fluorescence detection of labeled dsRNAs to enhance the sensitivity of sedimentation velocity measurements and directly characterize the interaction of PKR with duplex RNAs ranging from 20-40 bp and at multiple salt concentrations. The binding affinities increase at lower salt concentration and with longer dsRNA length, and in some cases the stoichiometries of binding of PKR to dsRNA increase at 75 mM NaCl. The dependence of the binding stoichiometries on dsRNA length does not conform to standard statistical models for nonspecific binding. We propose a model where the activation of PKR by longer RNAs is correlated with an alternative binding mode that allows for direct interaction of the kinase domains.