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.

Potential Alu function: regulation of the activity of double-stranded RNA-activated kinase PKR.

Cell stress, viral infection, and translational inhibition increase the abundance of human Alu RNA, suggesting that the level of these transcripts is sensitive to the translational state of the cell. To determine whether Alu RNA functions in translational homeostasis, we investigated its role in the regulation of double-stranded RNA-activated kinase PKR. We found that overexpression of Alu RNA by cotransient transfection increased the expression of a reporter construct, which is consistent with an inhibitory effect on PKR. Alu RNA formed stable, discrete complexes with PKR in vitro, bound PKR in vivo, and antagonized PKR activation both in vitro and in vivo. Alu RNAs produced by either overexpression or exposure of cells to heat shock bound PKR, whereas transiently overexpressed Alu RNA antagonized virus-induced activation of PKR in vivo. Cycloheximide treatment of cells decreased PKR activity, coincident with an increase in Alu RNA. These observations suggest that the increased levels of Alu RNAs caused by cellular exposure to different stresses regulate protein synthesis by antagonizing PKR activation. This provides a functional role for mammalian short interspersed elements, prototypical junk DNA.

Characterization of the solution complex between the interferon-induced, double-stranded RNA-activated protein kinase and HIV-I trans-activating region RNA.

The antiviral activity of the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase (PKR) is mediated through dsRNA binding leading to PKR autophosphorylation and subsequent inhibition of protein synthesis. Previous biochemical studies have suggested that autophosphorylation of PKR occurs via a protein-protein interaction and that PKR can form dimers in vitro. Using four independent biophysical and biochemical methods, we have characterized the solution complex formed between PKR and trans-activating region (TAR) RNA, a 57-nucleotide RNA species with double-stranded secondary structure derived from the human immunodeficiency virus type I genome. Chemical cross-linking and gel filtration analyses of PKR.TAR RNA complexes reveals that TAR RNA addition increases PKR dimerization and results in the formation of a solution complex with a molecular weight of approximately 150,000. Addition of TAR RNA to PKR results in a quenching of tryptophan fluorescence, indicative of a conformational shift. Through small angle neutron scattering analysis, we show that PKR exists in solution predominantly as a dimer, and has an elongated solution structure. Addition of TAR RNA to PKR causes a significant conformational shift in the protein at a 2:1 stoichiometric ratio of protein to RNA. Taken together, these data indicate that the PKR activation complex consists of a protein dimer bound cooperatively to one dsRNA molecule.

Mutational analysis of the double-stranded RNA (dsRNA) binding domain of the dsRNA-activated protein kinase, PKR.

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase, PKR, is an inhibitor of translation and has antiviral, antiproliferative, and antitumor properties. Previously, the dsRNA binding domain had been located within the N-terminal region of PKR and subsequently shown to include two nearly identical domains comprising residues 55-75 and 145-166. We have undertaken both random and site-directed, alanine-scanning mutagenesis in order to investigate the contribution of individual amino acids within these domains to dsRNA binding. Here we identify 2 residues that were absolutely required for dsRNA binding, glycine 57 and lysine 60. Mutation of 2 other residues within the domain (lysine 64 and leucine 75) resulted in less than 10% binding (compared to wild type). We have also identified a number of other residues that influence dsRNA binding to varying degrees. Mutants that were unable to bind dsRNA were not active in vitro and possessed no antiproliferative activity in vivo. However, dsRNA binding mutants were partially transdominant over wild type PKR in mammalian cells, suggesting that binding of dsRNA activator is not the mechanism responsible for the phenotype of PKR mutants.

The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin.

The mechanism by which bacterial heat-stable enterotoxins (ST I STA) cause diarrhea in humans and animals has been linked to the activation of an intestinal membrane-bound guanylate cyclase. Guanylin, a recently discovered rat intestinal peptide, is homologous in structure to ST I and can activate guanylate cyclase present on the human colonic carcinoma cell line T84. To directly test the mechanistic association of guanylate cyclase activation with diarrhea, we synthesized guanylin and a guanylin analog termed N9P10 guanylin and compared their biological activities with those of a synthetic ST I analog, termed ST Ib(6-18). We report that guanylin is able to inhibit the binding of a radiolabeled ST I analog to rat intestinal cells but causes diarrhea in infant mice only at doses at least 4 orders of magnitude higher than that of ST Ib(6-18). In contrast, N9P10 guanylin was enterotoxic in mice at much lower doses than guanylin but proved to be a weaker inhibitor of radiolabeled ST I than guanylin in the receptor binding assay. The pattern of guanylate cyclase activation observed for ST Ib(6-18) and the two guanylin analogs parallels the results observed in the receptor binding assay rather than those observed in the diarrheal assay. Treatment of guanylin with chymotrypsin or lumenal fluid derived from newborn mouse intestines resulted in a rapid loss of binding activity. Together, these results suggest that ST I enterotoxins may represent a class of long-lived superagonists of guanylin.

Structural characterization of functionally important regions of the Escherichia coli heat-stable enterotoxin STIb.

The biological properties of the Escherichia coli enterotoxin STIb (STA-3, STh) reside in a 13 amino acid C-terminal domain, abbreviated STIb(6-18). This tridecapeptide contains six cysteine residues involved in three intramolecular disulfide bridges. The solution structure of STIb(6-18) has been modeled as a series of three consecutive reverse turns [Gariépy et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 83, 8907-8911]. Synthetic tridecapeptide analogues of STIb(6-18) with single amino acid substitutions at non-cysteine sites, as well as a truncated decapeptide lacking one of the three disulfide bridges, were prepared in order to examine the relationship between primary sequence and biological activity. The relative affinity of each analogue for intestinal cell receptors only partially correlates with their dose-dependent ability to cause diarrhea in suckling mice, suggesting that subsaturation doses of the enterotoxin with respect to receptor occupancy on intestinal cells may be sufficient to cause diarrhea. Two substitutions in the central-turn region of the molecule, namely, Asn12----Ala and Ala14----D-Ala, resulted in a large decrease or loss of receptor binding activity as compared to native STIb(6-18), pointing out the functional importance of this region. Analogues containing replacements at other sites showed moderate to slight reductions in biological activity. In particular, residues in the C-terminal region appear to be less important for activity, although their presence remains essential, since a truncated analogue missing the last three amino acids is inactive.(ABSTRACT TRUNCATED AT 250 WORDS)