Mechanism of interaction of the double-stranded RNA (dsRNA) binding domain of protein kinase R with short dsRNA sequences.
The dsRNA-activated protein kinase (PKR) plays a major role in the cellular response to viral infection. PKR contains an N-terminal dsRNA binding domain (dsRBD) and a C-terminal kinase domain. The dsRBD consists of two tandem copies of a conserved double-stranded RNA binding motif, dsRBM1 and dsRBM2. dsRNA binding is believed to activate PKR by inducing dimerization and subsequent autophosphorylation reactions. We have characterized the function of the dsRBD by assessing the binding of dsRBM1 and dsRBD to a series of dsRNA sequences ranging from 15 to 45 bp. For dsRBM1, the binding stoichiometries agree with an overlapping ligand binding model where the motif binds to multiple faces of the dsRNA duplex and overlaps along the helical axis. Similar behavior is observed for a dsRBD containing both dsRBM1 and dsRBM2 for sequences up to 30 bp; however, the binding affinity is enhanced 30-fold. Longer dsRNA sequences exhibit lower-than-expected stoichiometries, indicating a change in binding mode. NMR spectroscopy was used to define the regions of the dsRBD that interact with dsRNA. dsRNA binding induces exchange broadening of cross-peaks in 1H-15N HSQC spectra. For a 20 bp dsRNA, the resonances most affected map to the known dsRNA binding regions of dsRBM1 as well as the N-terminus of dsRBM2. For a longer 40 bp sequence, additional regions of dsRBM2 exhibit enhanced broadening. These data support a model in which dsRBM1 plays the dominant role in binding short dsRNA sequences and dsRBM2 makes additional interactions with the longer sequences capable of activating PKR.
Ucci
Jason W
JW
Department of Molecular and Cell Biology and National Analytical Ultracentrifugation Facility, University of Connecticut, Storrs, Connecticut 06269-3125, USA.
Kobayashi
Yumiko
Y
Choi
Gregory
G
Alexandrescu
Andrei T
AT
Cole
James L
JL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000737:chemistry* / Q000378:metabolism*
D012330:RNA, Double-Stranded; D019892:eIF-2 Kinase