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J Biol Chem. 2008 July 18; 283(29): 20361–20371. doi: 10.1074/jbc.M803225200. | PMCID: PMC2459268 |
Copyright © 2008, The American Society for Biochemistry and
Molecular Biology, Inc. A Novel Family of Sequence-specific Endoribonucleases Associated with the
Clustered Regularly Interspaced Short Palindromic
Repeats *![[S with combining enclosing square]](/corehtml/pmc/pmcents/x0053x20DE.gif) Natalia Beloglazova, ‡§ Greg Brown, ‡§ Matthew D. Zimmerman, ¶ Michael Proudfoot, ‡§ Kira S. Makarova, ** Marina Kudritska, ‡§ Samvel Kochinyan, ‡§ Shuren Wang, ¶ Maksymilian Chruszcz, ¶ Wladek Minor, ¶ Eugene V. Koonin, **1 Aled M. Edwards, ‡§ Alexei Savchenko, ‡§ and Alexander F. Yakunin ‡§2‡Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada, §Structural Proteomics in Toronto, Ontario Cancer Institute, Max Bell Research Centre 5R407, Toronto, Ontario M5G 2C4, Canada, the ¶Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908, the Midwest Center for Structural Genomics, Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439, and the **National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894 Received April 28, 2008; Revised May 14, 2008. Clustered regularly interspaced short palindromic repeats (CRISPRs)
together with the associated CAS proteins protect microbial cells from
invasion by foreign genetic elements using presently unknown molecular
mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting
that these uncharacterized proteins play a central role in this process. Here
we show that the CAS2 proteins represent a novel family of endoribonucleases.
Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs
preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404
from Sulfolobus solfataricus, cleaved the phosphodiester linkage on
the 3′-side and generated 5′-phosphate- and
3′-hydroxyl-terminated oligonucleotides. The crystal structure of
SSO1404 was solved at 1.6Å resolution revealing the first ribonuclease
with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues
(Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for
enzymatic activity and suggested that Asp-10 might be the principal catalytic
residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we
propose that their role in the CRISPR-mediated anti-phage defense might
involve degradation of phage or cellular mRNAs. Numerous prokaryotic genomes contain structures known as clustered
regularly interspaced short palindromic repeats
(CRISPRs), 3 composed
of 25-50-bp direct repeats separated by intervening sequence spacers (or
inserts) of similar length
( 1- 3).
CRISPRs represent the most widely distributed family of repeats in prokaryotes
and are found in a great variety of bacteria and archaea, including many
pathogens (such as Mycobacterium tuberculosis, Streptococcus, Yersinia,
Neisseria, and Corynebacterium)
( 4- 8).
Most organisms possess multiple CRISPR clusters
( 2- 20)
that form tandem arrays containing from 4 to over 100 units (a repeat and a
spacer) ( 1,
9- 11).
Sequence analysis of the CRISPR spacers in various organisms revealed that
some of the spacer sequences are homologous (up to 100% sequence identity) to
sequences in phage genomes, plasmids, or in the chromosome and have either
sense or antisense orientations
( 5,
12- 14).
Remarkably, phages and plasmids fail to infect the specific strains carrying
the cognate spacers, implying a relationship between CRISPRs and immunity
against specific foreign DNA. Recent experiments on Streptococcus
thermophilus have directly demonstrated that, after a phage challenge,
this bacterium adds new CRISPR spacers derived from the phage genome
( 15,
16). Removal or addition of
the phage-specific spacers correlated with the cell resistance to the
respective phage implying that resistance specificity is determined by the
sequence identity between the phage genome and spacers
( 15). On the chromosome, CRISPR loci are flanked by a large number of
cas (CRISPR-associated) genes encoding uncharacterized proteins. A
comprehensive bioinformatic analysis of the CAS system in sequenced genomes
resulted in a refined classification with 25 gene families and at least nine
types of the cas operon organization
( 9,
17). Eight CAS protein
families have been predicted to possess nuclease activity; nine families have
been characterized as putative RNA-binding proteins (RAMP-domain proteins),
and two families have been predicted to possess helicase and DNA/RNA
polymerase activity ( 17). This
analysis, combined with the data on the homology of some CRISPR spacer
sequences to phage genes, led to the hypothesis that the CRISPRs and
cas-encoded proteins comprise a system of defense against invading
phages and plasmids and that this system might operate analogously to the
eukaryotic RNA interference systems
( 17). The wide distribution of
the CRISPR-CAS system among archaea and bacteria, its apparent importance for
immunity of prokaryotes against infectious agents, and the predicted novel
mechanism have recently made this system a subject of intense interest
( 3). Two cas genes ( cas1 and cas2) are always located
near a CRISPR locus and are found only in species containing CRISPRs
suggesting that these proteins play a central role in the CRISPR system
( 1,
17). The members of the CAS2
superfamily are small, uncharacterized proteins (80-120 residues), which
belong to COG1343 and COG3512 groups of the COG protein classification system
( 18). CAS2 proteins contain
several conserved sequence motifs, in particular an N-terminal motif that
consists of a string of hydrophobic residues (a predicted β-strand), and
typically ends with an aspartate
( 17). The CAS2 protein
sequences show some similarity to the sequences of the VapD family of
uncharacterized proteins that are functionally linked to the VapBC
toxin-antitoxin (TA) operon
( 19). Based on the pattern of
conserved amino acid residues, in particular the presence of a conserved
aspartate after a predicted β-strand, and some functional clues on the TA
systems, it has been hypothesized that both CAS2 and VapD might possess RNase
activity ( 17). Here we report for the first time the results of biochemical and structural
characterization of a family of CRISPR-associated enzymes, the CAS2 family
proteins from five prokaryotes. We show that CAS2 proteins are
endoribonucleases that are specific to single-stranded (ss)RNAs and
preferentially cleave them within U-rich regions. The crystal structure of a
representative CAS2 protein, SSO1404 from Sulfolobus solfataricus,
was solved to a 1.6 Å resolution and revealed a ferredoxin-like fold
with the double split β-α-β motif, as well as the putative
active site. Protein Overexpression, Purification, and Site-directed
Mutagenesis—The cloning of the genes encoding SSO1404 and other
CAS2 proteins (SSO8090, TM1796, AF1876, MTH1083, and NE0845) into the modified
pET15b was carried out as described previously
( 20). The proteins were
expressed as a fusion with an N-terminal His 6 tag in
Escherichia coli strain BL21 (DE3) and purified to more than 95%
homogeneity using metal-chelate affinity chromatography on nickel affinity
resin and gel filtration on a Superdex 200 26/60 column (Amersham Biosciences)
as described before ( 20,
21). Site-directed mutagenesis
of SSO1404 was performed as described previously
( 21) using a protocol based on
the QuikChange site-directed mutagenesis kit (Stratagene). Preparation of RNA Substrates—The short RNA substrates
( Table 1) were purchased from
IDT. The oligonucleotides were 5′-end-labeled with
[γ- 32P]ATP (6,000 Ci/mmol; Amersham Biosciences) and T4
polynucleotide kinase (PNK) (Fermentas) and then purified by denaturing PAGE
(15% polyacrylamide, 8 m urea gel). The labeled oligonucleotides
were eluted from the gel, precipitated with 2% LiClO 4 in acetone,
washed with acetone, dried, and dissolved in diethyl pyrocarbonate-treated
Milli-Q water. The long RNA substrates were synthesized using the Ambion T7
RNA polymerase MAXIscript transcription kit. For the synthesis of the mouse
β-actin mRNA (304 nt) fragment, pTRI-actin-mouse DNA (MAXIscript kit,
Ambion) was used as a template. To produce long CRISPR RNA substrates, a DNA
template was prepared by cloning the 1,292-bp-long 5′-fragment of the
S. solfataricus CRISPR cluster-2 DNA into the pBluescript SK+ vector
(Stratagene). During the transcription reaction (MAXIscript kit, Ambion), this
DNA template (linearized by EcoRV) directed the synthesis of a 270-nt-long RNA
substrate containing the CRISPR cluster-2 upstream region, repeat-1, spacer-1,
repeat-2, and a 27-nt fragment of spacer-2. Long RNA substrates were uniformly
labeled during transcription using [α- 32P]UTP (3,000
Ci/mmol). The labeled RNA substrates were purified by denaturing 8% PAA, 8
m urea gel, eluted, ethanol-precipitated, and stored at -70 °C.
Double-stranded RNA substrates were prepared by the incubation of equimolar
amounts (10 μ m) of the 5′- 32P-labeled sense and
unlabeled antisense RNA oligoribonucleotides (RNA5, RNA25, and RNA26) in 50
m m Tris-HCl (pH 7.0), 100 m m KCl at 90 °C for 1 min
and slow cooling at room temperature for 1.5 h. The formation of RNA duplexes
was verified by electrophoresis in 10% nondenaturing PAA gels. | TABLE 1Nucleotide sequences of ssRNA substrates and SSO1404 cleavage
sites |
Enzymatic Assays—The reaction mixture for RNase assays (10
μl) contained 0.1 μ m [ 32P]RNA, 50 m m
Tris-HCl (pH 8.5), 100 m m KCl, 5 m m MgCl 2, 1
m m dithiothreitol, and 0.01-0.1 μg of enzyme. The pH dependence
of SSO1404 was characterized using three buffers: MES-K (pH 5.5 to 6.5),
Tris-HCl (pH 7.0 to 9.0), and CAPS-K (pH 9.4 to 11.0). The reaction mixture
was incubated at 37 °C for the indicated period of time and quenched by
the addition of equal volume of formamide loading buffer (80% formamide,
0.025% bromphenol blue, 0.025% xylene cyanol, and 10 m m EDTA (pH
8.0)). The reaction products were resolved by electrophoresis in 15% PAA, 8
m urea gels using TBE (10 m m Tris borate (pH 8.3), and 2
m m EDTA) as a running buffer. As nucleotide size markers, an
imidazole ladder or a G-ladder produced by partial RNA cleavage by 2
m imidazole or RNase T1, respectively, was used
( 22,
23). For the analysis of the
RNA product 5′-end, after RNase reaction, RNA products were precipitated
by 2% LiClO 4, washed by acetone, dried, dissolved in Milli-Q water,
and phosphorylated with [γ- 32P]ATP and T4 PNK using
conditions for forward or phosphate exchange reaction according to the
manufacturer's protocol (Fermentas). After the PNK reaction, RNA products were
analyzed using denaturing 15% PAA, 8 m urea gels as described above
for RNase assays. The reaction mixtures for DNase assays (40 μl) contained
50 m m HEPES-K buffer (pH 7.5), 100 m m KCl, 5
m m MgCl 2, 1 m m dithiothreitol, 0.3 μg of
λ DNA (double-stranded DNA) or 0.75 μg of M13 DNA (ssDNA), and 1-4
μg of enzyme. After 1 h of incubation at 37 °C, the reactions were
quenched by the addition of 6× DNA loading dye (Fermentas) and analyzed
on EtBr-stained 1% agarose gels. Protein Crystallization and Structure Determination—SSO1404
crystals were grown using the hanging drop vapor diffusion method with the
drops containing a mixture of 2 μl of 10 mg/ml purified
selenomethionine-incorporated SSO1404 protein and 2 μl of reservoir buffer
(0.2 m NaI, 20% w/v PEG 3350, and 2% v/v isopropyl alcohol). For
diffraction studies, the crystals were stabilized with the crystallization
buffer supplemented with 20% ethylene glycol as a cryoprotectant and
flash-frozen in liquid nitrogen. A single crystal of
selenomethionine-incorporated SSO1404 was used to collect diffraction data at
beamline 19-BM of the Structural Biology Center of the Advanced Photon Source
( 24) and was maintained at a
temperature of 100 K. A single-wavelength anomalous diffraction dataset was
collected at a wavelength of 0.9794 Å. Crystallographic data collection
and model refinement statistics are summarized in
Table 2. Reflection data were
collected, indexed, integrated, and scaled with HKL-3000
( 25). | TABLE 2Crystallographic data collection and refinement statistics |
A two-site selenium substructure was determined; the structure was phased
by single-wavelength anomalous diffraction, and an initial model was built.
All structure solution and initial model building was performed by HKL-3000,
which is integrated with SHELXD, SHELXE, MLPHARE, DM, O, COOT, SOLVE, RESOLVE,
and ARP/wARP
( 25- 32).
The initial model was improved by iterative cycles of manual rebuilding in
COOT, followed by maximum likelihood refinement with REF-MAC5
( 33). In later stages of
refinement, a multigroup TLS model generated by the TLSMD web server was used
to further improve the model. The final model was validated using Mol-probity
( 34), SFCHECK
( 35), and PROCHECK. The atomic
coordinates and structure factors for SSO1404 have been deposited in the
Protein Data Bank with the accession code 2i8e. Enzymatic Activity of CAS2 Proteins—To characterize the
biochemical activity of CAS2 proteins, we cloned and purified six members of
this family from different organisms as follows: SSO1404 and SSO8090 from
S. solfataricus, AF1876 from Archaeoglobus fulgidus, TM1796
from Thermotoga maritima, MTH1083 from Methanobacterium
thermoautotrophicum, and NE0845 from Nitrosomonas europaea.
Given the prediction that CAS2 proteins might possess nuclease activity
( 17), the purified proteins
were tested for the presence of DNase or RNase activity against
single-stranded (ss) DNA (M13 DNA), double-stranded (ds) DNA (λ DNA),
or ssRNA as follows: RNA5, RNA6, and RNA14
( Table 1), which are identical
in sequence to the sense strands of the CRISPR repeats or spacers from S.
solfataricus, A. fulgidus, or T. maritima (36 or 39 nt). No
nuclease activity was found against either of the DNA substrates, but all
proteins degraded the ssRNAs (). With the tested ssRNA substrates, the CAS2 proteins generated
a limited number (one to five) of products of various lengths (7-29 nt)
indicating that they cleave ssRNAs endonucleolytically. A similar but not
identical pattern of products was observed, and most cleavage sites contained
one or two U (). This
observation suggests that the CAS2 ribonucleases recognize similar RNA
sequences but also display some difference in substrate preference. SSO1404
also showed detectable cleavage of a long model RNA substrate (the 304-nt
transcript of the 5′-fragment of the mouse β-actin gene) and
generated several products of various lengths (18-200 nt), but the activity
was lower than that against short oligoribonucleotides (data not shown). Thus,
the CAS2 family proteins are ssRNA-specific endoribonucleases. Reaction Requirements and RNA Cleavage Products of
SSO1404—SSO1404 exhibited RNase activity over a broad pH range
(7.0-10.0) with maximum activity at pH 8.5-9.0
(). Very little
cleavage of RNA5 by SSO1404 (between U20 and C21) was observed in the absence
of both monovalent and divalent cations, as well as in the presence of
Na+, K+, or Mn2+
(). Cleavage
between U9 and U10 was greatly enhanced by the addition of Mg2+.
The Mg2+-dependent activity was further stimulated by
K+, whereas Na+ had inhibitory effect. Thus, SSO1404
requires Mg2+ (5 mm), K+ (100 mm),
and pH 8.5-9.0 for optimal cleavage of ssRNA. RNases can be subdivided into two groups depending on the position of the
cleavage of the phosphodiester linkage
( 36). Enzymes of the first
class cleave the bond on the 3′-side (producing a
5′-phosphorylated second product) and include numerous intracellular
endo- and exoribonucleases. The enzymes of the second class ( e.g.
RNase A, RNase T1, and barnases) cleave the linkage on the 5′-side
releasing products containing a cyclic 2′,3′-phosphodiester bond.
The products of ssRNA cleavage by a representative CAS2 protein, SSO1404, were
characterized using the T4 PNK-catalyzed reactions of RNA phosphorylation (at
5′-hydroxyl termini) and the phosphate exchange between the
5′-phosphate groups of the oligonucleotide substrate and ATP
( 37). After hydrolysis of the
RNA5 substrate by SSO1404, the reaction products were incubated with PNK and
[γ- 32P]ATP and analyzed by denaturing PAGE and
autoradiography (). Incubation with PNK did not produce a labeled
3′-end product (no second band on the gel), and the gel showed only one
labeled band corresponding to the 5′-end product
(, lane
2). This suggests that the 3′-end product is phosphorylated on its
5′-end. The presence of this 3′-end product in the reaction
mixture with SSO1404 was confirmed using the PNK-catalyzed phosphate exchange
reaction between the RNA 5′-phosphate and [γ- 32P]ATP in
the presence of ADP excess
( 38). This experiment produced
two labeled products on the gel with the expected length of 9 and 30 nt
(, lane
3). Thus, SSO1404 and apparently other CAS2 enzymes are the
5′-phosphomonoester-producing endoribonucleases. Endoribonuclease Activity of SSO1404 against Long CRISPR
Substrates—Previous analyses of small RNAs produced in S.
solfataricus and A. fulgidus revealed that the long CRISPR
transcripts are processed into fragments with the size of one repeat and one
spacer (62-75 nt) ( 39,
40). It has been proposed that
these short CRISPR RNAs are produced by an unknown cas RNase that
would cleave within the repeat sequence producing intact spacers flanked with
the repeat fragments ( 3). The
demonstration of ssRNase activity of SSO1404 suggested the possibility that
CAS2 could be the RNase that is responsible for processing CRISPR RNAs. Thus,
we tested SSO1404 for the ability to cleave the 270-nucleotide-long CRISPR
transcript of the S. solfataricus CRISPR cluster-2. The uniformly
labeled long CRISPR RNA substrate was prepared using T7 RNA polymerase and the
270-bp DNA fragment of the 5′-end of the S. solfataricus CRISPR
cluster-2 containing the upstream region, repeat-1, spacer-1, repeat-2, and a
27-nt fragment of spacer-2 as the template. If SSO1404 were a
CRISPR-processing endoribonuclease, it would be expected to cleave this
substrate within the repeat sequences and produce a minimal product containing
one spacer flanked by two repeat halves (63-66-nt-long), as well as a series
of products containing two or more repeat + spacer units. SSO1404 showed
detectable endoribonuclease activity with this long CRISPR RNA substrate, but
it produced products that were smaller than expected (23, 27, and 45 nt long),
as well as several longer fragments (65, 75, 95, 110, and 160 nt long)
(). This
finding indicates that SSO1404 does not have the expected specificity toward
CRISPR transcripts and thus is unlikely to be the CRISPR-processing
endoribonuclease. SSO1404 Cleaves ssRNAs Preferentially within U-rich
Regions—The substrate specificity of SSO1404 was further
characterized using an extended set of shorter synthetic ssRNA substrates, as
well as several dsRNAs. The RNA1 to RNA5 substrates were identical in sequence
to the sense strands of repeat-1 (24 nt), repeat-2 (25 nt), and three spacers
( 1- 3)
of the S. solfataricus CRISPR cluster-2
( Table 1). SSO1404 cleaved all
these substrates, but the highest activity was observed with RNA5, which was
preferentially cleaved between U9 and U10
(). The
hydrolysis of this substrate proceeded to near completion with the formation
of one main labeled product. Eight ssRNA substrates (RNA7 to RNA10 and RNA14 to RNA17) corresponded to
repeats and spacers from other CRISPR-containing organisms ( S.
thermophilus, T. maritima, and Methanococcus jannaschii),
whereas RNA11, RNA12, and RNA13 were scrambled RNA substrates
( Table 1). SSO1404 demonstrated
endonucleolytic activity toward most of these substrates with the highest
activity toward RNA9, RNA10, and RNA13
(). The
analysis of the cleavage sites showed that SSO1404 preferred U-rich sequences,
and in many substrates ( e.g. RNA9, RNA10, RNA12, and RNA13) the
cleavage site was located between two Us
( Table 1). Examination of the
computer-predicted secondary structures of the cleavable RNA substrates
revealed that SSO1404 cleaves RNA substrates in predicted single-stranded
regions (). No cleavage
was observed with the dsRNA substrates prepared by annealing ssRNA substrates
(RNA5, RNA25, and RNA26) with their complementary RNA chains and containing
blunt ends or a two-nucleotide 5′-overhang (not shown). Thus, SSO1404 is
a single-stranded RNA-specific endoribonuclease. Although several substrates shown in
(RNA2, RNA5, and RNA12)
contain a potential SSO1404 cleavage site within the predicted loops, the
enzyme cleaved these substrates only in close proximity to the 5′- or
3′-ends suggesting that it requires the presence of a free RNA end. To
determine whether a stem-loop is required as an entry site for SSO1404, we
tested its activity against three truncated oligoribonucleotides (16 nt) that
lack the potential to form stem-loops and contain the SSO1404 cleavage site
from RNA5 (RNA18), RNA11 (RNA19), or RNA9 (RNA20). SSO1404 efficiently cleaved
all three substrates at the same position as in the corresponding longer
substrates ( Table 1) indicating
that the primary sequences of these short oligoribonucleotides were sufficient
to direct site-specific cleavage by SSO1404, and no specific secondary
structure was required. To determine the minimal size of the SSO1404
substrate, the 39-nt-long RNA5 was further trimmed to 16-nt RNA18, 13-nt
RNA21, 10-nt RNA22, 8-nt RNA23, and 6-nt RNA24
( Table 1). SSO1404 efficiently
cleaved the four longer substrates (RNA5, RNA18, RNA21, and RNA22) but showed
no activity against RNA23 (8 nt) and RNA24 (6 nt)
(). Thus,
oligoribonucleotides as short as 10 nt can serve as SSO1404 substrates. An ssRNA endoribonuclease activity with a preference for U-rich regions
also has been observed in the abortive phage infection determinant AbiB from
Lactococcus lactis and in mRNA interferases MazF-mt1 and MazF-mt6
from M. tuberculosis
( 41,
42). The L. lactis
AbiB prevented growth of the sensitive phage bIL170 through the selective
degradation of phage mRNAs by endonucleolytic cleavage at U/U, A/U, and U/A
sites ( 41). The mRNA
interferases are toxin components of chromosomal TA modules that are abundant
in free-living prokaryotes and induce reversible cell cycle arrest or
programmed cell death in response to starvation or other stress conditions
( 43- 46).
These enzymes have different mRNA cleavage specificities, and their expression
in the cell causes the effective inhibition of protein synthesis leading to
temporal cell growth arrest. In this regard, it might be relevant that U-rich
and AU-rich regions have been identified upstream of the Shine-Dalgarno
sequence in prokaryotic and phage mRNAs and shown to enhance translation
( 47,
48). The CAS2 proteins are
unrelated to mRNA interferases or AbiB but, as mentioned above, appear to be
homologous to VapD, a component of a distinct class of TA systems. Thus, CAS2
and VapD might represent a novel group of mRNA-specific endoribonucleases. Crystal Structure of SSO1404—To elucidate the molecular
basis for the ssRNase activity of CAS2, we determined the crystal structure of
SSO1404 at 1.6 Å resolution. The structure demonstrated that SSO1404 is
a homodimer ().
This is consistent with the results of our gel filtration experiments, which
showed that the native protein has a molecular mass of 23.7 ± 0.6 kDa
(predicted molecular mass 11.9 kDa). In the SSO1404 dimer, two monomers are
joined by their β-sheets, and the α-helices are exposed on the
surface. In the SSO1404 monomer, the four β-strands form an antiparallel
β-sheet, and the α-helices are packed on one side, and the overall
structure may be described as a double-split β-α-β fold. In
addition, the last β-strand (β5) of each monomer interacts with the
β-sheet of the other monomer creating two-joint, five-strand,
antiparallel β-sheets (). A search for structural homologs of SSO1404 using the Dali
( 49) and secondary-structure
matching ( 50) programs
(Pro-Func data base ( 51)
identified two uncharacterized proteins, TT1823 from Thermus
thermophilus (Protein Data Bank code 1zpw, secondary-structure matching
Z-score = 7.7, r.m.s.d. = 1.3 Å) and PF1117 from Pyrococcus
furiosus (Protein Data Bank code 2i0x, secondary-structure matching
Z-score = 7.0, r.m.s.d. = 1.7 Å)
(). Like
SSO1404, these two proteins belong to the CAS2 family (COG1343)
( 17). In addition, the Dali
search recognized structures of domain III of the translation elongation
factor G (EF-G) from T. thermophilus (Protein Data Bank code 1fnm)
and elongation factor 2 from yeast (Protein Data Bank code 1n0v,
Z-score = 6.1, r.m.s.d. = 2.3 Å) as significantly similar to
SSO1404. All these structures have the double-split β-α-β
fold, which is also observed in many ribosomal and other RNA-binding proteins
( 52). However, the ribosomal
proteins and elongation factors do not have the conserved residues that are
important for the RNase activity of SSO1404 (Tyr-9, Asp-10, Arg-17, Arg-19,
Arg-31, and Phe-37) (see below). Both Dali and VAST searches also recognized various proteins with the
ferredoxin-like fold as being structural relatives of each of the three CAS2
structures with similar, moderate scores and r.m.s.d. values (supplemental
Table 1), suggesting that CAS2 proteins comprise a new superfamily within the
ferredoxin-like fold. Indeed, this is how the available CAS2 structures have
been classified in the SCOP data base. The ferredoxin-like domains consist of one of the most populated protein
folds, with numerous structural and functional derivatives
( 53). In particular, this fold
is present in numerous RNA-binding proteins, including the RNA-binding domain,
the anticodon-binding domain of PheRS, ribosomal proteins S6 and S10, and also
the prominent components of the CAS system, the RAMP superfamily proteins,
which possess a duplicated ferredoxin-like domain
( 17,
54). However, to our
knowledge, no nucleases with the ferredoxin-like fold have been characterized
with the possible exception of the IS200 transposase of S.
solfataricus (SSO1474, Protein Data Bank code 2f5g). Like many ribosomal
proteins ( 55), SSO1404 exposes
several aromatic and hydrophobic residues to the solvent (Ile-32, Tyr-34,
Ile-75, Ile-84, Val-85, Ile-86, Phe-80, and Tyr-88), and some of these
residues are conserved in large subsets of the CAS2 family proteins
(supplemental Fig. 1), suggesting their involvement in the interaction with
the bases of RNA. The extended loop α2-β4 of SSO1404 also could be
a candidate for interaction with RNA. Mutational Analysis of SSO1404 and the Potential Catalytic
Site—The structure of the SSO1404 dimer revealed several major
cavities or grooves located at the interface of the two monomers, which might
represent potential catalytic sites (). The most prominent cavity consists of the deep cleft
between two monomers accommodating the side chains of conserved Asp-10, which
is bracketed by two α2-β4 loops
(). To identify
the active site residues involved in RNA cleavage, we performed alanine
replacement mutagenesis of the conserved residues of SSO1404. A multiple
sequence alignment of CAS2 proteins
( 17) identified several
conserved amino acids (;
supplemental Fig. 1). The single conserved aspartate, Asp-10, was of special
interest as the putative principal catalytic residue. In the SSO1404
structure, Asp-10 is located at the end of the first β-strand and is
preceded by the conserved tyrosine Tyr-9. Several other partially conserved
residues are spatially close to Asp-10, including Arg-17, Arg-19, and Arg-31.
Alanine replacement mutagenesis of SSO1404 revealed that Asp-10, Arg-31, and
Phe-37 are strictly required for the catalytic activity, whereas Y9A, R17A,
and R19A showed very low activity (). The T12A and D65A mutants showed an ~2-fold drop in
activity, whereas the other mutant proteins exhibited slightly reduced (Q33A,
Y34A, and S35A) or wild-type level (D13A, D14A, N18A, and R67A) activity. In the SSO1404 structure, five residues that are important for activity
(Tyr-9, Asp-10, Arg-17, Arg-31, and Phe-37) are located in the long cavity
formed by the α1 helix on one side, the β2 and β3 strands on
the other side, and the β1 strand at the bottom
(). SSO1404
requires a divalent metal cation for activity, Mg 2+ being the
optimal metal (). In most known RNases, two or three conserved acidic
residues are critical for catalysis and are involved in the coordination of
one or two metal cations (Mg 2+ or Mn 2+), which activate
a nucleophilic water molecule for hydrolysis of the phosphodiester bond or
stabilize the transition state in cleavage reactions
( 56). The structures of
SSO1404 and two other CAS2 proteins (TT1823 and PF1117) revealed no metal
atoms bound to the protein. Nevertheless, given that SSO1404 required the
addition of Mg 2+ or Mn 2+ for activity and the conserved
Asp-10 was critical for catalysis, we suggest that this residue is involved in
the coordination of the metal cation in SSO1404 and other CAS2 proteins.
Interestingly, the side chains of Asp-10 in the two monomers are oriented
toward the active site of the other monomer and are positioned 6.5 Å
apart ()
suggesting that these residues do not form two independent active sites. In
addition, these aspartates are located inside a deep, narrow cleft making them
inaccessible for direct interaction with the RNA substrate
(). Therefore,
we propose that the side chains of the two Asp-10 bind one Mg 2+
atom, which might coordinate a nucleophilic water molecule. As shown here
(), SSO1404
generates products terminating in a 3′-hydroxyl and a 5′-phosphate
suggesting that, like in the E. coli RNase E, the reaction involves
an attack of a water molecule on the susceptible phosphodiester bond followed
by scission of the 3′-oxygen-phosphorus bond
( 57). Therefore, the catalytic
mechanism of SSO1404 probably involves the in-line nucleophilic attack on the
scissile phosphate by an activated hydroxyl group generated from a water
molecule that is coordinated to magnesium. The SSO1404 D65A protein had a 2-fold reduced activity
(). This conserved
aspartate is located on the long α2-β4 loop
() that varies in length among CAS2 proteins and
accommodates several partially conserved charged residues (Glu-64, Asp-65,
Glu-66, and Arg-67 in SSO1404) (). Based on the structure of the SSO1404 dimer
(), we suggest that the α2-β4 loops are
responsible for the recognition of RNA substrates and might determine the
substrate selectivity of different CAS2 proteins. We propose that like the
E. coli RNase E S1 domain
( 58), the SSO1404
α2-β4 loop might function to clamp down the RNA substrate in the
active site orienting its phosphate backbone for nucleophilic attack by the
magnesium-coordinated activated water molecule. Conclusions—This work is the first step toward a
comprehensive biochemical and structural characterization of CAS proteins.
Recent bioinformatic analyses suggest that the CRISPR and CAS proteins might
use several dissimilar mechanisms to abrogate phage infection and that at
least one of these mechanisms could resemble eukaryotic RNA interference
( 3,
17). This study shows that
SSO1404 and other CAS2 proteins represent a novel family of endoribonucleases,
which possess a ferredoxin-like fold and are specific to ssRNA substrates.
SSO1404 cleaves ssRNAs preferentially within U-rich regions and, in this
respect, resembles the phage abortive infection endoribonuclease AbiB from
L. lactis ( 41). A
similar mechanism of selective degradation of phage transcripts might be
proposed for SSO1404 and other CAS2 proteins as well. Under this hypothesis,
CAS2 would be the functional analog of the eukaryotic slicer nuclease, a
function that is performed by the PIWI domains that are unrelated to CAS2
( 59). Considering the apparent
mechanism of CRISPR-associated anti-phage defense that involves integration of
sequences homologous to fragments of phage mRNAs into the CRISPR loci, the
CAS2 endoribonuclease activity might contribute to this process as well.
Another possible role of CAS2 proteins in anti-phage defense might be
associated with the global inhibition of translation by mRNA cleavage, a
mechanism that has been proposed for RelBE and several other TA systems
(MazEF, PemIK, and ChpBIK) that contain RNase components (mRNA interferases)
( 45,
60,
61). These TA systems play
important roles in stress response to nutritional limitations or DNA damage,
and their expression results in growth arrest or programmed cell death
( 43- 45).
Moreover, it has been shown that the E. coli MazEF TA module prevents
multiplication of the phage P1 by promoting cell death
( 62). A similar mechanism of
anti-phage response appears to be a possibility for CAS2, especially in light
of the previously described relationship with VapD, an uncharacterized protein
that is functionally linked to the VapBC toxin-antitoxin system
( 17). S. solfataricus
and many other CRISPR-containing organisms have at least two CAS2 proteins
that might have different sequence specificities and could target distinct
sets of mRNAs. We thank all members of the Structural Proteomics in Toronto (SPiT) Centre,
the Structural Biology Center at the Advanced Photon Source and Midwest Center
for Structural Genomics, and Andrzej Joachimiak for help in conducting
experiments and discussions. Crystallography results were derived from work
performed at Argonne National Laboratory, Structural Biology Center, at the
Advanced Photon Source. Argonne is operated by University of Chicago Argonne,
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