Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans
| Autor: | Dennis G. Cvitkovitch, Natalia Beloglazova, Kemin Tan, Xiaohui Xu, Hong Cui, M. Anca Serbanescu, Alexander F. Yakunin, Milosz Ruszkowski, Alexei Savchenko, Anna N. Khusnutdinova, Sofia Lemak, Greg Brown, Andrzej Joachimiak |
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| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular SeMet selenomethionine crystal structure XorCas5c Cas5c from Xanthomonas oryzae Protein Data Bank (RCSB PDB) Biochemistry DvuCas5c Cas5c from Desulfovibrio vulgaris Streptococcus mutans 03 medical and health sciences Ribonucleases 0302 clinical medicine Bacterial Proteins PDB Protein Data Bank CFU colony-forming unit Cas5c Catalytic triad SpyCas5c Cas5c from Streptococcus pyogenes Cas5 CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Ribonuclease RNA Processing Post-Transcriptional Cas6 BhaCas5c Cas5c from Bacillus halodurans Site-directed mutagenesis Molecular Biology PNK T4 polynucleotide kinase 030304 developmental biology Trans-activating crRNA 0303 health sciences biology RNA recognition motif Chemistry SmuCas5c Cas5c from Streptococcus mutans RNA RAMP repeat-associated mysterious protein Cell Biology R–S–R repeat–spacer–repeat RNA Bacterial RRM RNA recognition motif biology.protein CRISPR-Cas Systems ribonuclease site-directed mutagenesis crRNA CRISPR-RNA 030217 neurology & neurosurgery Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| DOI: | 10.1016/j.jbc.2021.101251 |
| Popis: | 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 3′-base of the stem but shows significant tolerance to substrate sequence variations downstream of the cleavage site. Structure-based 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. |
| Databáze: | OpenAIRE |
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