Molecular basis for PKR activation by PACT or dsRNA

*Department of Molecular Genetics and
^‡Structural Biology Program and Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195; and
^†Graduate Program in Molecular Virology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved May 17, 2006 (received for review March 21, 2006)

Abstract

The mammalian protein kinase PKR is a critical component of the innate immune response against virus infection. Its cellular actions are mediated by modulating cell signaling and translational regulation. To be enzymatically active, latent PKR needs to be activated by binding to one of its activators, dsRNA or PACT protein. Although the structures of the N-terminal dsRNA-binding domain and the C-terminal kinase domain of PKR have been separately determined, the mode of activation of the enzyme remains unknown. To address this problem, we used biochemical, genetic, and NMR analyses to identify the PACT-binding motif (PBM) located in the kinase domain and demonstrated an intramolecular interaction between PBM and dsRNA-binding domain. This interaction is responsible for keeping PKR in an inactive conformation, because its disruption by point mutations of appropriate residues produced constitutively active PKR. Furthermore, a short decoy peptide, representing PBM, was able to activate PKR by interfering with the intramolecular interaction. These observations suggest a model for PKR activation upon binding of dsRNA or PACT.

Footnotes

^§To whom correspondence should be addressed at:
Department of Molecular Genetics/NE20, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195.
E-mail: seng{at}ccf.org
Author contributions: S.L., G.A.P., J.Q., and G.C.S. designed research; S.L., G.A.P., K.D., and X.Z. performed research; S.L., G.A.P., K.D., J.Q., and G.C.S. analyzed data; and S.L., G.A.P., J.Q., and G.C.S. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

dsRBD,

dsRNA-binding domain;

PBM,

PACT-binding motif;

KD,

kinase domain;

MBP,

maltose-binding protein;

NOE,

nuclear Overhauser effect.