TY - JOUR

T1 - Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans

AU - Lemak, Sofia

AU - Serbanescu, M. Anca

AU - Khusnutdinova, Anna N.

AU - Ruszkowski, Milosz

AU - Beloglazova, Natalia

AU - Xu, Xiaohui

AU - Brown, Greg

AU - Cui, Hong

AU - Tan, Kemin

AU - Joachimiak, Andrzej

AU - Cvitkovitch, Dennis G.

AU - Savchenko, Alexei

AU - Yakunin, Alexander

PY - 2021/11

Y1 - 2021/11

N2 - The cariogenic pathogen Streptococcus mutans contains twoCRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNAtranscripts (pre-crRNA) containing repeats and spacers tomature crRNA guides. In this study, we determined the crystalstructure of SmuCas5c at a resolution of 1.72 Å, which revealedthe presence of an N-terminal modified RNA recognition motifand a C-terminal twisted β-sheet domain with four boundsulphate molecules. Analysis of surface charge and residueconservation of the SmuCas5c structure suggested the locationof an RNA-binding site in a shallow groove formed by the RNArecognition motif domain with several conserved positivelycharged residues (Arg39, Lys52, Arg109, Arg127, and Arg134).Purified SmuCas5c exhibited metal-independent ribonucleaseactivity against single-stranded pre-CRISPR RNAs containing astem–loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within therepeat sequence at a single cleavage site located at the 30-baseof the stem but shows significant tolerance to substratesequence variations downstream of the cleavage site. Structurebased mutational analysis revealed that the conserved residuesTyr50, Lys120, and His121 comprise the SmuCas5c catalyticresidues. In addition, site-directed mutagenesis of positivelycharged residues Lys52, Arg109, and Arg134 located near thecatalytic triad had strong negative effects on the RNase activityof this protein, suggesting that these residues are involved inRNA binding. Taken together, our results reveal functionaldiversity of Cas5c ribonucleases and provide further insightinto the molecular mechanisms of substrate selectivity andactivity of these enzymes.

AB - The cariogenic pathogen Streptococcus mutans contains twoCRISPR systems (type I-C and type II-A) with the Cas5c protein (SmuCas5c) involved in processing of long CRISPR RNAtranscripts (pre-crRNA) containing repeats and spacers tomature crRNA guides. In this study, we determined the crystalstructure of SmuCas5c at a resolution of 1.72 Å, which revealedthe presence of an N-terminal modified RNA recognition motifand a C-terminal twisted β-sheet domain with four boundsulphate molecules. Analysis of surface charge and residueconservation of the SmuCas5c structure suggested the locationof an RNA-binding site in a shallow groove formed by the RNArecognition motif domain with several conserved positivelycharged residues (Arg39, Lys52, Arg109, Arg127, and Arg134).Purified SmuCas5c exhibited metal-independent ribonucleaseactivity against single-stranded pre-CRISPR RNAs containing astem–loop structure with a seven-nucleotide stem and a pentaloop. We found SmuCas5c cleaves substrate RNA within therepeat sequence at a single cleavage site located at the 30-baseof the stem but shows significant tolerance to substratesequence variations downstream of the cleavage site. Structurebased mutational analysis revealed that the conserved residuesTyr50, Lys120, and His121 comprise the SmuCas5c catalyticresidues. In addition, site-directed mutagenesis of positivelycharged residues Lys52, Arg109, and Arg134 located near thecatalytic triad had strong negative effects on the RNase activityof this protein, suggesting that these residues are involved inRNA binding. Taken together, our results reveal functionaldiversity of Cas5c ribonucleases and provide further insightinto the molecular mechanisms of substrate selectivity andactivity of these enzymes.

U2 - 10.1016/j.jbc.2021.101251

DO - 10.1016/j.jbc.2021.101251

M3 - Article

C2 - 34592310

VL - 297

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 5

M1 - 101251

ER -