Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation.

Protein kinase PKR is an interferon-induced enzyme that plays a key role in the control of viral infections and cellular homeostasis. Compared with other known kinases, PKR is activated by a distinct mechanism that involves double-stranded RNA (dsRNA) binding in its N-terminal region in an RNA sequence-independent fashion. We report here the solution structure of the 20 kDa dsRNA-binding domain (dsRBD) of human PKR, which provides the first three-dimensional insight into the mechanism of its dsRNA-mediated activation. The structure of dsRBD exhibits a dumb-bell shape comprising two tandem linked dsRNA-binding motifs (dsRBMs) both with an alpha-beta-beta-beta-alpha fold. The structure, combined with previous mutational and biochemical data, reveals a highly conserved RNA-binding site on each dsRBM and suggests a novel mode of protein-RNA recognition. The central linker is highly flexible, which may enable the two dsRBMs to wrap around the RNA duplex for cooperative and high-affinity binding, leading to the overall change of PKR conformation and its activation.     

trans-Autophosphorylation by the isolated kinase domain is not sufficient for dimerization or activation of the dsRNA-activated protein kinase PKR

The double-stranded (ds) RNA-activated protein kinase PKR phosphorylates the alpha-subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits translation initiation. PKR contains two dsRNA binding domains in its amino terminus and a kinase domain in its carboxy terminus. dsRNA binding activates PKR from a latent state by inducing dimerization and trans-autophosphorylation. Recent studies show that PKR is also activated by caspase cleavage to remove the inhibitory dsRNA binding domains. In this report, we show that the isolated kinase domain of PKR is a constitutively active monomeric kinase that has an activity similar to that of wild-type PKR. We used a solid-phase kinase assay system to show that PKR does not transfer its own phosphate to either PKR or eIF2alpha but rather uses the gamma-phosphate from ATP. In addition, the isolated autophosphorylated kinase domain of PKR phosphorylated intact monomeric PKR in trans in a reaction that did not require dsRNA binding. However, this trans-phosphorylation did not occur at the critical Thr446/451 sites and was not sufficient to induce dimerization and/or activation of PKR. The results show that dsRNA binding domains of PKR are not only required for dimerization of PKR but also required for phosphorylation of Thr446/451 sites of PKR. The results imply that even though the isolated kinase domain of PKR phosphorylates intact PKR and eIF2alpha, it is unable to activate PKR.     

Regulation of PKR by HCV IRES RNA: Importance of Domain II and NS5A

Protein kinase R (PKR) is an essential component of the innate immune response. In the presence of double-stranded RNA (dsRNA), PKR is autophosphorylated, which enables it to phosphorylate its substrate, eukaryotic initiation factor 2α, leading to translation cessation. Typical activators of PKR are long dsRNAs produced during viral infection, although certain other RNAs can also activate. A recent study indicated that full-length internal ribosome entry site (IRES), present in the 5′-untranslated region of hepatitis C virus (HCV) RNA, inhibits PKR, while another showed that it activates. We show here that both activation and inhibition by full-length IRES are possible. The HCV IRES has a complex secondary structure comprising four domains. While it has been demonstrated that domains III-IV activate PKR, we report here that domain II of the IRES also potently activates. Structure mapping and mutational analysis of domain II indicate that while the double-stranded regions of the RNA are important for activation, loop regions contribute as well. Structural comparison reveals that domain II has multiple, non-Watson-Crick features that mimic A-form dsRNA. The canonical and noncanonical features of domain II cumulate to a total of ∼ 33 unbranched base pairs, the minimum length of dsRNA required for PKR activation. These results provide further insight into the structural basis of PKR activation by a diverse array of RNA structural motifs that deviate from the long helical stretches found in traditional PKR activators. Activation of PKR by domain II of the HCV IRES has implications for the innate immune response when the other domains of the IRES may be inaccessible. We also study the ability of the HCV nonstructural protein 5A (NS5A) to bind various domains of the IRES and alter activation. A model is presented for how domain II of the IRES and NS5A operate to control host and viral translation during HCV infection.     

Characterization of the direct physical interaction of nc886, a cellular non-coding RNA, and PKR

We have recently shown that nc886 (pre-miR-886 or vtRNA2-1) is not a genuine microRNA precursor nor a vault RNA, but a novel type of non-coding RNA that represses PKR, a double-stranded RNA (dsRNA) dependent kinase. Here we have characterized their direct physical association. PKR’s two RNA binding domains form a specific and stable complex with nc886’s central portion, without any preference to its 5′-end structure. By binding to PKR with a comparable affinity, nc886 competes with dsRNA and attenuates PKR activation by dsRNA. Our data suggest that nc886 sets a threshold for PKR activation so that it occurs only during genuine viral infection but not by a minute level of fortuitous cellular dsRNA.     

The N-terminus of PKR is responsible for the activation of the NF-kappaB signaling pathway by interacting with the IKK complex

The interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) has been shown to activate NF-kappaB independently of its kinase function after interaction with the IKK complex. In order to investigate the mechanism of NF-kappaB activation by PKR, we identified the domain of PKR responsible for stimulating the NF-kappaB pathway in PKR-deficient fibroblasts using an NF-kappaB dependent reporter assay. The N-terminal 1-265 AA of PKR activates NF-kappaB, whereas the 1-180 AA N-terminus restricted to the two dsRNA Binding Domains (DRBD), the third basic domain alone (AA 181-265), or the C-terminus of PKR (AA 266-550) were unable to stimulate the expression of the NF-kappaB dependent reporter gene. Using confocal microscopy, we confirmed that PKR full length as well as PKR N-terminus colocalized with IKKbeta. By GST-pulldown analysis, using different PKR domains, we then revealed the specific ability of the PKR N-terminus 1-265 to bind to and activate IKK and showed that this activation requires the integrity of the IKK complex. This activation is not only due to DRBDs since the DRBD fragment 1-180 failed to inhibit PKR 1-265 induced NF-kappaB activation. Our results therefore demonstrate that the ability of PKR to mediate NF-kappaB activation resides in its full N-terminus, and requires both DRBDs and the third basic domain.     

A dynamically tuned double-stranded RNA binding mechanism for the activation of antiviral kinase PKR

A key step in the activation of interferon-inducible antiviral kinase PKR involves differential binding of viral double-stranded RNA (dsRNA) to its two structurally similar N-terminal dsRNA binding motifs, dsRBM1 and dsRBM2. We show here, using NMR spectroscopy, that dsRBM1 with higher RNA binding activity exhibits significant motional flexibility on a millisecond timescale as compared with dsRBM2 with lower RNA binding activity. We further show that dsRBM2, but not dsRBM1, specifically interacts with the C-terminal kinase domain. These results suggest a dynamically tuned dsRNA binding mechanism for PKR activation, where motionally more flexible dsRBM1 anchors to dsRNA, thereby inducing a cooperative RNA binding for dsRBM2 to expose the kinase domain.     

Domain stabilities in protein kinase R (PKR): evidence for weak interdomain interactions

PKR (protein kinase R) is induced by interferon and is a key component of the innate immunity antiviral pathway. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase, leading it to phosphorylate eIF2alpha, thus inhibiting protein synthesis in virally infected cells. PKR contains a dsRNA-binding domain (dsRBD) and a kinase domain. The dsRBD is composed of two tandem dsRNA-binding motifs. An autoinhibition model for PKR has been proposed, whereby dsRNA binding activates the enzyme by inducing a conformational change that relieves the latent enzyme of the inhibition that is mediated by the interaction of the dsRBD with the kinase. However, recent biophysical data support an open conformation for the latent enzyme, where activation is mediated by dimerization of PKR induced upon binding dsRNA. We have probed the importance of interdomain contacts by comparing the relative stabilities of isolated domains with the same domain in the context of the intact enzyme using equilibrium chemical denaturation experiments. The two dsRNA-binding motifs fold independently, with the C-terminal motif exhibiting greater stability. The kinase domain is stabilized by about 1.5 kcal/mol in the context of the holenzyme, and we detect low-affinity binding of the kinase and dsRBD constructs in solution, indicating that these domains interact weakly. Limited proteolysis measurements confirm the expected domain boundaries and reveal that the activation loop in the kinase is accessible to cleavage and unstructured. Autophosphorylation induces a conformation change that blocks proteolysis of the activation loop.     

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.     

Double-stranded RNA deaminase ADAR1 increases host susceptibility to virus infection

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the alpha subunit of eukaryotic initiation factor 2 (eIF-2alpha) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR+/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR-/- cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.     

Specific recognition of HIV TAR RNA by the dsRNA binding domains (dsRBD1-dsRBD2) of PKR

PKR (double-stranded RNA-dependent protein kinase) is an important component of host defense to virus infection. Binding of dsRNA to two dsRBDs (double-stranded RNA binding domains) of PKR modulates its own kinase activation. How structural features of natural target RNAs, such as bulges and loops, have an effect on the binding to two dsRBDs of PKR still remains unclear. By using ITC and NMR, we show here that both the bulge and loop of TAR RNA are necessary for the high affinity binding to dsRBD1-dsRBD2 of PKR with 1:1 stoichiometry. The binding site for the dsRBD1-dsRBD2 spans from upper bulge to lower stem of the TAR RNA, based on chemical shift mapping. The backbone resonances in the 40 kDa TAR.dsRBD1-dsRBD2 were assigned. NMR chemical shift perturbation data suggest that the beta1-beta2 loop of the dsRBD1 interacts with the TAR RNA, whereas that of the dsRBD2 is less involved in the TAR RNA recognition. In addition, the residues of the interdomain linker between the dsRBD1 and the dsRBD2 also show large chemical perturbations indicating that the linker is involved in the recognition of TAR RNA. The results presented here provide the biophysical and spectroscopic basis for high-resolution structural studies, and show how local RNA structural features modulate recognition by dsRBDs.     

Recognition of viral RNA stem–loops by the tandem double-stranded RNA binding domains of PKR

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem–loops; HIV-1 TAR and adenovirus VA_I-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA–protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.     

Activation of the protein kinase PKR by short double-stranded RNAs with single-stranded tails

The human RNA-activated protein kinase PKR is an interferon-induced protein that is part of the innate immune response and inhibits viral replication. The action of PKR involves RNA-dependent autophosphorylation leading to inhibition of translation. PKR has an N-terminal dsRNA-binding domain that can interact non-sequence specifically with long (>33 bp) stretches of dsRNA leading to activation. In addition, certain viral and cellular RNAs containing non-Watson-Crick structures and multiple, shorter dsRNA sections can regulate PKR. In an effort to identify novel binders and possible activators of PKR, we carried out selections on a partially structured dsRNA library using truncated and full-length versions of PKR. A library with 10(11) sequences was constructed and aptamers that bound to His6-tagged proteins were isolated. Characterization revealed a novel minimal RNA motif for activation of PKR with the following unified structural characteristics: a hairpin with a nonconserved imperfect 16-bp dsRNA stem flanked by 10-15-nt single-stranded tails, herein termed a "ss-dsRNA motif." Boundary experiments revealed that the single-stranded tails flanking the dsRNA core provide the critical determinant for activation. The ss-dsRNA motif occurs in a variety of cellular and viral RNAs, suggesting possible novel functions for PKR in nature.     

Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2α), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2α in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2α phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

Activation of PKR: an open and shut case?

The double-stranded (ds) RNA-activated protein kinase, PKR, has a key role in the innate immunity response to viral infection in higher eukaryotes. PKR contains an N-terminal dsRNA-binding domain and a C-terminal kinase domain. In the prevalent autoinhibition model for PKR activation, dsRNA binding induces a conformational change that leads to the release of the dsRNA-binding domain from the kinase, thus relieving the inhibition of the latent enzyme. Structural and biophysical data now favor a model whereby dsRNA principally functions to induce dimerization of PKR via the kinase domain. This dimerization model has implications for other PKR regulatory mechanisms and represents a new structural paradigm for control of protein kinase activity.