Heparin Activates PKR by Inducing Dimerization

Abstract

The RNA activated kinase PKR plays a key role in the innate immunity response to viral infection. PKR is induced by interferon in a latent form that is activated by binding double stranded RNA (dsRNA) or RNAs that contain duplex regions to undergo autophosphorylation. Activation by dsRNA is mediated by PKR dimerization. PKR can also be activated by heparin, a highly sulfated glycosaminoglycan. PKR activation by heparin does not require the presence of the dsRNA binding domain and heparin does not compete with dsRNA for binding to PKR, indicating that heparin and dsRNA bind at different sites. We have characterized the mechanism of PKR activation by heparin oligosaccharides of defined sizes. The smallest heparin capable of robust PKR activation is the hexasaccharide (dp6). PKR binding affinity is strongly dependent on both the heparin length and ionic strength. Dissociation constants measured in 75 mM NaCl vary from ∼230 nM for dp8 to 32 uM for dp2. Sedimentation velocity measurements using interference and fluorescence detection indicate that dp8 binds to monomeric PKR and also increases PKR dimerization. The velocity data fit well to a classical linkage model where heparin binding is linked with PKR self-association with a coupling free energy of ΔG= −0.80 kcal/mole. Thus, both dsRNA and heparin activate PKR by enhancing dimerization.