Distinct Z-DNA binding mode of a PKR-like protein kinase containing a Z-DNA binding domain (PKZ)

Abstract

Double-stranded ribonucleic acid-activated protein kinase (PKR) downregulates translation as a defense mechanism against viral infection. In fish species, PKZ, a PKR-like protein kinase containing left-handed deoxyribonucleic acid (Z-DNA) binding domains, performs a similar role in the antiviral response. To understand the role of PKZ in Z-DNA recognition and innate immune response, we performed structural and functional studies of the Z-DNA binding domain (Z alpha) of PKZ from Carassius auratus (caZ alpha(PKZ)). The 1.7-angstrom resolution crystal structure of caZ alpha(PKZ):Z-DNA revealed that caZ alpha(PKZ) shares the overall fold with other Z alpha, but has discrete structural features that differentiate its DNA binding mode from others. Functional analyses of caZ alpha(PKZ) and its mutants revealed that caZ alpha(PKZ) mediates the fastest B-to-Z transition of DNA among Z alpha, and the minimal interaction for Z-DNA recognition is mediated by three backbone phosphates and six residues of caZ alpha(PKZ). Structure-based mutagenesis and B-to-Z transition assays confirmed that Lys56 located in the beta-wing contributes to its fast B-to-Z transition kinetics. Investigation of the DNA binding kinetics of caZ alpha(PKZ) further revealed that the B-to-Z transition rate is positively correlated with the association rate constant. Taking these results together, we conclude that the positive charge in the beta-wing largely affects fast B-to-Z transition activity by enhancing the DNA binding rate.