Characterization of the Solution Complex between the Interferon-induced, Double-stranded RNA-activated Protein Kinase and HIV-I Trans-activating Region RNA

Abstract

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 PKR⅐TAR 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.

PKR; a sentinel kinase for cellular stress

“…DsRNA binding causes a major conformational change in PKR as evidenced by gel analyses of protein-RNA complexes (Manche et al, 1992), or by biophysical techniques using tryptophan¯uorescence quenching and neutron scattering (Carpick et al, 1997). The conformational change likely serves to uncover a catalytic site(s) for autophosphorylation, or to shift domains from di erent parts of the same PKR molecule (or from di erent PKR molecules within a protein-protein complex) into an active conformation.…”

“…The conformational change likely serves to uncover a catalytic site(s) for autophosphorylation, or to shift domains from di erent parts of the same PKR molecule (or from di erent PKR molecules within a protein-protein complex) into an active conformation. The ®nding that dsRNA binding causes PKR to elongate rather than contract (Carpick et al, 1997) suggests a mechanism, whereby the dsRBD would swing away from the rest of the protein, exposing the catalytic site. The second-order kinetics of PKR autophosphorylation suggests this reaction is intermolecular (Kostura and Mathews, 1989).…”

“…The second-order kinetics of PKR autophosphorylation suggests this reaction is intermolecular (Kostura and Mathews, 1989). In fact dimerization is important for kinase activation (Langland and Jacobs, 1992;Thomis and Samuel, 1993;Romano et al, 1995;Patel et al, 1995;Ortega et al, 1996;Kaufman, 1996, 1997;Carpick et al, 1997;Tan et al, 1998) although exact molecular mechanisms and the protein structural and dsRNA requirements for dimerization, remain to be determined. PKR mutants which are capable of dimerization but lack dsRNA binding activity are biologically inactive, indicating that dimerization alone is not su cient for PKR activity (Patel et al, 1995(Patel et al, , 1996Wu and Kaufman, 1996).…”

“…PKR mutants which are capable of dimerization but lack dsRNA binding activity are biologically inactive, indicating that dimerization alone is not su cient for PKR activity (Patel et al, 1995(Patel et al, , 1996Wu and Kaufman, 1996). Moreover, the inactive mutant PKR (K296R, Figure 1a) can dimerize in vitro (Carpick et al, 1997). PKR dimerization may be mediated in part through the dsRBD, either through direct protein-protein interactions in this region (Patel et al, 1995;, or through dsRNA bridging the protein subunits (Cosentino et al, 1995; but domains within the C-terminal half of PKR are also required and dimerization (Romano et al, 1995;Tan et al, 1998) can be blocked by a peptide corresponding to amino acids 244±296 (Tan et al, 1998).…”

“…An autoradiograph of samples analysed by SDS-polyacrylamide gel electrophoresis is shown N-terminal 280 amino acids of PKR do not interact with the full length protein (Ortega et al, 1996). Highly puri®ed PKR can form dimers in the absence of dsRNA, and is in equilibrium between the monomeric and dimeric forms (Carpick et al, 1997). Addition of dsRNA tends to shift this equilibrium towards the dimer (Carpick et al, 1997), either through a bridging e ect or through a protein conformational change (or both).…”