Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing - PubMed (original) (raw)

Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing

Judith Reeks et al. Biochem J. 2013.

Abstract

The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed 'CRISPR' (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unit-length crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes.

PubMed Disclaimer

Figures

Figure 1

Figure 1. SsoCas6 cleaves a CRISPR RNA repeat

(A) Representative time course of RNA cleavage by SsoCas6 at 60°C under single-turnover conditions, analysed by gel electrophoresis and phosphorimaging. Labels ‘s’ and ‘p’ indicate substrates and products respectively. The control reaction ‘c’ was carried out in the absence of SsoCas6 for 25 min. (B) Single-turnover kinetic rates for cleavage of a CRISPR repeat RNA by SsoCas6 as a function of reaction temperature. The sequence of the RNA oligonucleotide substrate is shown at the top with the cleavage position indicated with an arrow. Each rate was calculated from at least six data points as described in the Experimental section, with means±S.E.M. calculated from curve fitting shown.

Figure 2

Figure 2. The crystal structure of SsoCas6

(A) Schematic representation of a typical ferredoxin-like fold with the β-strands as blue arrows and the α-helices as cyan cylinders. The N- and C-termini are shown as blue and red spheres respectively. (B) The structure of SsoCas6 with secondary-structure elements labelled. Disordered loops are shown as broken black lines and the glycine-rich loop shown in yellow. The location of the missing α-helix of the N-terminal domain is indicated. (C) View of the SsoCas6 dimer. (D) Electrostatic surface potential of the SsoCas6 dimer generated using CCP4MG [50]. The black boxes indicate the active-site region, with the broken box indicating the active site on the non-visible face of the dimer.

Figure 3

Figure 3. Dimerization of SsoCas6

(A) View of Leu170 and Val202 at the dimer interface. These residues were each changed to aspartate to disrupt the interface. (B) Gel-filtration elution profiles of dimeric SsoCas6 and the SsoCas6-L170D/V202D variant, which elutes with a retention volume consistent with a monomeric composition (expected molecular mass of 33 kDa). (C) Thermofluor analysis of heat-induced denaturation of wild-type (WT) and monomeric SsoCas6, showing the 7°C difference in melting temperatures (Tm). (D) Single-turnover kinetic comparison of wild-type and monomeric SsoCas6. The catalytic activity of the monomer is reduced by 95% compared with the wild-type (WT) enzyme.

Figure 4

Figure 4. Delineating the SsoCas6 active site

(A) Phosphorimage of a denaturing polyacrylamide gel showing the reaction products of repeat RNA incubated with wild-type (WT) and selected variant Cas6 enzymes. Lane m shows an RNA ladder generated by alkaline hydrolysis and lane c shows RNA incubated for 60 min in the absence of protein. Time points correspond to 1, 5 and 50 min incubations at 60°C. The cleavage site is indicated in the RNA sequence below the image. (B) Plot of the reaction kinetics for selected variants of SsoCas6 (red, wild-type, WT; black, S268A; green, K51A; blue, K28A). All data points were measured in triplicate and are means±S.E.M. (C) Structure of the SsoCas6 monomer. The glycine-rich loop is shown in yellow to highlight the approximate position of the active site. The positions of selected side chains targeted by site-directed mutagenesis are shown as magenta sticks. Of these residues, it was not possible to define the absolute conformation of the side chains of Lys25, Lys28 and Lys51 from the electron density. Chains B and D are superimposed on chain A to include all of the desired residues. (D) First-order rate constants for wild-type and selected variant SsoCas6 enzymes. Relative activity (Rel. act.) is expressed as a percentage of wild-type (WT) activity.

Similar articles

Cited by

References

    1. Mojica F. J., Diez-Villasenor C., Garcia-Martinez J., Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 2005;60:174–182. - PubMed
    1. Bolotin A., Quinquis B., Sorokin A., Ehrlich S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005;151:2551–2561. - PubMed
    1. Carte J., Wang R., Li H., Terns R. M., Terns M. P. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 2008;22:3489–3496. - PMC - PubMed
    1. Brouns S. J., Jore M. M., Lundgren M., Westra E. R., Slijkhuis R. J., Snijders A. P., Dickman M. J., Makarova K. S., Koonin E. V., van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science. 2008;321:960–964. - PMC - PubMed
    1. Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011;471:602–607. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources