TY - JOUR
T1 - A Type III-B Cmr effector complex catalyzes the synthesis of cyclic oligoadenylate second messengers by cooperative substrate binding
AU - Han, Wenyuan
AU - Stella, Stefano
AU - Zhang, Yan
AU - Guo, Tong
AU - Sulek, Karolina
AU - Peng-Lundgren, Li
AU - Montoya, Guillermo
AU - She, Qunxin
N1 - Funding Information:
National Science Foundation of China [31771380]; Danish Council for Independent Research [DFF-4181-00274]; Novo Nordisk Foundation Center for Protein Research, Novo Nordisk Foundation [NNF14CC0001]. Funding for open access charge: National Science Foundation of China [31771380]. Conflict of interest statement. None declared.
Publisher Copyright:
© The Author(s) 2018.
PY - 2018
Y1 - 2018
N2 - Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α-RNP)was purified fromthe native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α-RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme-substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.
AB - Recently, Type III-A CRISPR-Cas systems were found to catalyze the synthesis of cyclic oligoadenylates (cOAs), a second messenger that specifically activates Csm6, a Cas accessory RNase and confers antiviral defense in bacteria. To test if III-B CRISPR-Cas systems could mediate a similar CRISPR signaling pathway, the Sulfolobus islandicus Cmr-α ribonucleoprotein complex (Cmr-α-RNP)was purified fromthe native host and tested for cOA synthesis. We found that the system showed a robust production of cyclic tetra-adenylate (c-A4), and that c-A4 functions as a second messenger to activate the III-B-associated RNase Csx1 by binding to its CRISPR-associated Rossmann Fold domain. Investigation of the kinetics of cOA synthesis revealed that Cmr-α-RNP displayed positively cooperative binding to the adenosine triphosphate (ATP) substrate. Furthermore, mutagenesis of conserved domains in Cmr2α confirmed that, while Palm 2 hosts the active site of cOA synthesis, Palm 1 domain serves as the primary site in the enzyme-substrate interaction. Together, our data suggest that the two Palm domains cooperatively interact with ATP molecules to achieve a robust cOA synthesis by the III-B CRISPR-Cas system.
UR - http://www.scopus.com/inward/record.url?scp=85056271765&partnerID=8YFLogxK
U2 - 10.1093/nar/gky844
DO - 10.1093/nar/gky844
M3 - Journal article
C2 - 30239876
AN - SCOPUS:85056271765
SN - 0305-1048
VL - 46
SP - 10319
EP - 10330
JO - Nucleic Acids Research
JF - Nucleic Acids Research
IS - 19
ER -