The cariogenic pathogen Streptococcus mutans contains two
CRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNA
transcripts (pre-crRNA) containing repeats and spacers to
mature crRNA guides. In this study, we determined the crystal
structure of SmuCas5c at a resolution of 1.72 Å, which revealed
the presence of an N-terminal modified RNA recognition motif
and a C-terminal twisted β-sheet domain with four bound
sulphate molecules. Analysis of surface charge and residue
conservation of the SmuCas5c structure suggested the location
of an RNA-binding site in a shallow groove formed by the RNA
recognition motif domain with several conserved positively
charged residues (Arg39, Lys52, Arg109, Arg127, and Arg134).
Purified SmuCas5c exhibited metal-independent ribonuclease
activity against single-stranded pre-CRISPR RNAs containing a
stem–loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within the
repeat sequence at a single cleavage site located at the 30
-base
of the stem but shows significant tolerance to substrate
sequence variations downstream of the cleavage site. Structurebased mutational analysis revealed that the conserved residues
Tyr50, Lys120, and His121 comprise the SmuCas5c catalytic
residues. In addition, site-directed mutagenesis of positively
charged residues Lys52, Arg109, and Arg134 located near the
catalytic triad had strong negative effects on the RNase activity
of this protein, suggesting that these residues are involved in
RNA binding. Taken together, our results reveal functional
diversity of Cas5c ribonucleases and provide further insight
into the molecular mechanisms of substrate selectivity and
activity of these enzymes.