Molecular basis for cOA6 synthesis by a Type-IIIA CRISPR-Cas enzyme and its conversion to cOA4 production

Hemant N Goswami¹, Bing Wang¹, Fozieh Ahmadizadeh¹, Doreen Addo-Yobo², Yu Zhao¹, Arthur Willington³, Michael P. Terns⁴ & Hong Li^1,2

1 Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306, USA.

2 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, FL 32306, USA.

3 Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA

4 Biochemistry and Molecular Biology, Genetics and Microbiology, University of Georgia, Athens, GA, USA.

The Type III-A CRISPR-Cas systems (Csm) are multi-subunit and multipronged prokaryotic enzymes that initiate a cascade of neutralizing activities against viral invaders. In addition to cleaving the activating RNA transcripts, Csm confer two collateral activities: indiscriminately cleaving single-stranded DNA and synthesizing cyclic oligoadenylate (cOA) by the Cas10 subunit. Within the known Csm systems, Cas10 produces two major forms of the cOA molecules, cOA4 and cOA6, that are used to elicit cOA-dependent activities. Whereas the mechanism for cOA-dependent activation is well understood, the molecular basis for the synthesis of specific cOA molecules remains unclear. Here, we present structural characterization of a cOA6-producing Csm complex during its activation by a model target RNA. This analysis reveals three adenine binding sites characterized by conserved tyrosine-serine/threonine pairs. Comparing these sites with the known adenine binding sites in cOA4-producing Csm systems, both structurally and by sequence alignment, revealed a site specific to the cOA6-producing Csm systems. Consistently, disrupting this unique tyrosine-threonine pair specifically impaired cOA6 production while promoting cOA4 production. These findings suggest that while Cas10 utilizes a conserved enzymatic mechanism for forming the phosphodiester bond, it has evolved distinct strategies to regulate cOA chain length.