Dimeric structure of pseudokinase RNase L bound to 2-5A reveals a basis for interferon-induced antiviral activity.
RNase L is an ankyrin repeat domain-containing dual endoribonuclease-pseudokinase that is activated by unusual 2,'5'-oligoadenylate (2-5A) second messengers and which impedes viral infections in higher vertebrates. Despite its importance in interferon-regulated antiviral innate immunity, relatively little is known about its precise mechanism of action. Here we present a functional characterization of 2.5 Å and 3.25 Å X-ray crystal and small-angle X-ray scattering structures of RNase L bound to a natural 2-5A activator with and without ADP or the nonhydrolysable ATP mimetic AMP-PNP. These studies reveal how recognition of 2-5A through interactions with the ankyrin repeat domain and the pseudokinase domain, together with nucleotide binding, imposes a rigid intertwined dimer configuration that is essential for RNase catalytic and antiviral functions. The involvement of the pseudokinase domain of RNase L in 2-5A sensing, nucleotide binding, dimerization, and ribonuclease functions highlights the evolutionary adaptability of the eukaryotic protein kinase fold.
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
Hao
H
Huang
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Elton
E
Zeqiraj
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Beihua
B
Dong
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Babal Kant
BK
Jha
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Nicole M
NM
Duffy
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Stephen
S
Orlicky
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada.
Neroshan
N
Thevakumaran
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
Manisha
M
Talukdar
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada.
Monica C
MC
Pillon
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Derek F
DF
Ceccarelli
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
Leo C K
LC
Wan
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Yu-Chi
YC
Juang
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
Daniel Y L
DY
Mao
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA.
Christina
C
Gaughan
Department of Biology, Georgia State University, Atlanta, GA 30302, USA.
Margo A
MA
Brinton
Department of Biology, Georgia State University, Atlanta, GA 30302, USA.
Andrey A
AA
Perelygin
NE-CAT APS, Building 436E, Argonne National Lab, Argonne, IL 60439, USA.
Igor
I
Kourinov
Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4K1, Canada.
Alba
A
Guarné
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. Electronic address: silverr@ccf.org.
Robert H
RH
Silverman
Program in Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada; Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: sicheri@lunenfeld.ca.
Frank
F
Sicheri
D000227:Adenine Nucleotides / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry; D000266:Adenylyl Imidodiphosphate / Q000737:chemistry; D000818:Animals; D017089:Ankyrin Repeat; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D004686:Encephalomyocarditis virus; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009843:Oligoribonucleotides / Q000737:chemistry*; D010849:Picornaviridae; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship; D034421:Sus scrofa
D000227:Adenine Nucleotides; D009843:Oligoribonucleotides; D000266:Adenylyl Imidodiphosphate; C023505:2',5'-oligoadenylate; D000244:Adenosine Diphosphate; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease