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• W2010187816 abstract "Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3′ single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes. Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3′ single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes. Identification of Cas5d as pre-crRNA processor in type I-C/Dvulg CRISPR-Cas system Revealing the substrate specificity of in Cas5d endoribonuclease Identifying the catalytic triad in Cas5d via structure determination and mutagenesis Cas5d assembles into a Cascade-like complex with Csd1, Csd2, and crRNA Clustered regularly interspaced short palindromic repeats (CRISPRs) are found in about ∼45% of sequenced bacteria and ∼83% of archaea genomes and participate in RNA-based adaptive immunity against exogenous genetic elements, such as viruses (phages), invading conjugative plasmids, and transposable elements (Deveau et al., 2010Deveau H. Garneau J.E. Moineau S. CRISPR/Cas system and its role in phage-bacteria interactions.Annu. Rev. Microbiol. 2010; 64: 475-493Crossref PubMed Scopus (425) Google Scholar, Horvath and Barrangou, 2010Horvath P. Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea.Science. 2010; 327: 167-170Crossref PubMed Scopus (1579) Google Scholar, Karginov and Hannon, 2010Karginov F.V. Hannon G.J. The CRISPR system: small RNA-guided defense in bacteria and archaea.Mol. Cell. 2010; 37: 7-19Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, Marraffini and Sontheimer, 2010Marraffini L.A. Sontheimer E.J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea.Nat. Rev. Genet. 2010; 11: 181-190Crossref PubMed Scopus (711) Google Scholar, Sorek et al., 2008Sorek R. Kunin V. Hugenholtz P. CRISPR—a widespread system that provides acquired resistance against phages in bacteria and archaea.Nat. Rev. Microbiol. 2008; 6: 181-186Crossref PubMed Scopus (641) Google Scholar, van der Oost et al., 2009van der Oost J. Jore M.M. Westra E.R. Lundgren M. Brouns S.J. CRISPR-based adaptive and heritable immunity in prokaryotes.Trends Biochem. Sci. 2009; 34: 401-407Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, Waters and Storz, 2009Waters L.S. Storz G. Regulatory RNAs in bacteria.Cell. 2009; 136: 615-628Abstract Full Text Full Text PDF PubMed Scopus (1164) Google Scholar, Wiedenheft et al., 2012Wiedenheft B. Sternberg S.H. Doudna J.A. RNA-guided genetic silencing systems in bacteria and archaea.Nature. 2012; 482: 331-338Crossref PubMed Scopus (1305) Google Scholar). CRISPR loci are composed of invariable repeat sequences of about 21–48 base pairs (bp) in length, interspaced by variable spacer sequences (26–72 bp) derived from epichromosomal origins (Bolotin et al., 2005Bolotin A. Quinquis B. Sorokin A. Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.Microbiology. 2005; 151: 2551-2561Crossref PubMed Scopus (1119) Google Scholar, Mojica et al., 2005Mojica F.J. Díez-Villaseñor C. García-Martínez J. Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.J. Mol. Evol. 2005; 60: 174-182Crossref PubMed Scopus (1315) Google Scholar, Pourcel et al., 2005Pourcel C. Salvignol G. Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.Microbiology. 2005; 151: 653-663Crossref PubMed Scopus (902) Google Scholar). Adjacent to CRISPR loci is a cas (CRISPR associated) operon, encoding a cluster of Cas proteins involved in CRISPR interference and adaptation processes. The cas genes can be further classified into a set of “core” genes and subtype-specific genes based on phylogenetic analysis (Haft et al., 2005Haft D.H. Selengut J. Mongodin E.F. Nelson K.E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.PLoS Comput. Biol. 2005; 1: e60Crossref PubMed Scopus (737) Google Scholar, Makarova et al., 2006Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action.Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (834) Google Scholar, Makarova et al., 2011aMakarova K.S. Aravind L. Wolf Y.I. Koonin E.V. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems.Biol. Direct. 2011; 6: 38Crossref PubMed Scopus (336) Google Scholar, Makarova et al., 2011bMakarova K.S. Haft D.H. Barrangou R. Brouns S.J. Charpentier E. Horvath P. Moineau S. Mojica F.J. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (1655) Google Scholar). CRISPR-Cas systems are recently classified into three major types (type I, II, and III) based on the mutually exclusive presence of cas3, cas9/csn1, and cas10/cmr2 genes, respectively. Type I system is the most wide-spread and can be further classified into six different subtypes (I-A to I-F) based on the presence of distinct subtype-specific genes (Makarova et al., 2011bMakarova K.S. Haft D.H. Barrangou R. Brouns S.J. Charpentier E. Horvath P. Moineau S. Mojica F.J. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (1655) Google Scholar). At the molecular level, CRISPR interference can be divided into three stages: (1) CRISPR adaptation upon exposure to foreign genetic elements through the insertion of a new spacer into the 5′-end of the genomic CRISPR array; (2) expression of the nascent CRISPR RNA transcript (pre-crRNA) and enzymatic digestion into the mature form (crRNA); and (3) crRNA-mediated interference to degrade foreign genetic elements (Barrangou et al., 2007Barrangou R. Fremaux C. Deveau H. Richards M. Boyaval P. Moineau S. Romero D.A. Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (3829) Google Scholar, Brouns et al., 2008Brouns 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: 960-964Crossref PubMed Scopus (1668) Google Scholar, Carte et al., 2008Carte 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: 3489-3496Crossref PubMed Scopus (423) Google Scholar, Deltcheva et al., 2011Deltcheva 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: 602-607Crossref PubMed Scopus (1660) Google Scholar, Haft et al., 2005Haft D.H. Selengut J. Mongodin E.F. Nelson K.E. A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes.PLoS Comput. Biol. 2005; 1: e60Crossref PubMed Scopus (737) Google Scholar, Hale et al., 2009Hale C.R. Zhao P. Olson S. Duff M.O. Graveley B.R. Wells L. Terns R.M. Terns M.P. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.Cell. 2009; 139: 945-956Abstract Full Text Full Text PDF PubMed Scopus (771) Google Scholar, Haurwitz et al., 2010Haurwitz R.E. Jinek M. Wiedenheft B. Zhou K. Doudna J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease.Science. 2010; 329: 1355-1358Crossref PubMed Scopus (497) Google Scholar, Makarova et al., 2006Makarova K.S. Grishin N.V. Shabalina S.A. Wolf Y.I. Koonin E.V. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action.Biol. Direct. 2006; 1: 7Crossref PubMed Scopus (834) Google Scholar). The pre-crRNA processing in some CRISPR subtypes has been shown to be mediated by ferredoxin domain containing endoribonucleases encoded in the cas operon. These include Cse3 protein from subtype I-E (previously named Ecoli subtype), Csy4 from subtype I-F (Ypest), and Cas6 from subtype I-A (Apern), I-B (Tneap), and III-B (Cmr) have been shown to process the pre-crRNA in their corresponding CRISPR subtype (Brouns et al., 2008Brouns 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: 960-964Crossref PubMed Scopus (1668) Google Scholar, Carte et al., 2008Carte 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: 3489-3496Crossref PubMed Scopus (423) Google Scholar, Deltcheva et al., 2011Deltcheva 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: 602-607Crossref PubMed Scopus (1660) Google Scholar, Haurwitz et al., 2010Haurwitz R.E. Jinek M. Wiedenheft B. Zhou K. Doudna J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease.Science. 2010; 329: 1355-1358Crossref PubMed Scopus (497) Google Scholar). Structure and mechanistic diversity were found among these pre-crRNA processors. Cse3 and Csy4 recognize the palindromic RNA stemloop in pre-crRNAs in a site- and structure-specific manner (Gesner et al., 2011Gesner E.M. Schellenberg M.J. Garside E.L. George M.M. Macmillan A.M. Recognition and maturation of effector RNAs in a CRISPR interference pathway.Nat. Struct. Mol. Biol. 2011; 18: 688-692Crossref PubMed Scopus (134) Google Scholar, Haurwitz et al., 2010Haurwitz R.E. Jinek M. Wiedenheft B. Zhou K. Doudna J.A. Sequence- and structure-specific RNA processing by a CRISPR endonuclease.Science. 2010; 329: 1355-1358Crossref PubMed Scopus (497) Google Scholar, Sashital et al., 2011Sashital D.G. Jinek M. Doudna J.A. An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3.Nat. Struct. Mol. Biol. 2011; 18: 680-687Crossref PubMed Scopus (145) Google Scholar). Endoribonucleolytic cleavage proceeds through a general acid-base mechanism to generate a 5′ hydroxyl and a 2′,3′-cyclic phosphate. The stemloop portion of the mature crRNA was shown to remain associated with the endoribonuclease to participate in the formation of the bigger Cascade (CRISPR-associated complex for antiviral defense) complex. This complex was thought to display the crRNA for subsequent destruction of invading nucleic-acid sequences (Jore et al., 2011Jore M.M. Lundgren M. van Duijn E. Bultema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. et al.Structural basis for CRISPR RNA-guided DNA recognition by Cascade.Nat. Struct. Mol. Biol. 2011; 18: 529-536Crossref PubMed Scopus (420) Google Scholar, Wiedenheft et al., 2011Wiedenheft B. Lander G.C. Zhou K. Jore M.M. Brouns S.J. van der Oost J. Doudna J.A. Nogales E. Structures of the RNA-guided surveillance complex from a bacterial immune system.Nature. 2011; 477: 486-489Crossref PubMed Scopus (300) Google Scholar). Although using a similar enzymatic mechanism, Cas6 preferentially recognizes the nonpalindromic single stranded (ss-) RNA repeats and processes them using a “wrapping-mechanism” (Wang et al., 2011Wang R. Preamplume G. Terns M.P. Terns R.M. Li H. Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage.Structure. 2011; 19: 257-264Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). By contrast, the pre-crRNAs processing in the subtype II CRISPR-Cas systems uses a completely different mechanism involving Csn1 recognition and RNase III cleavage guided by the trans-encoded small RNAs (Deltcheva et al., 2011Deltcheva 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: 602-607Crossref PubMed Scopus (1660) Google Scholar). CRISPR interference has not been well studied in the subtype I-C CRISPR organisms. The molecular function of the three subtype I-C specific proteins Cas5d, Csd1, and Csd2 has not been reported. In this study, we show that pre-crRNA processing, the key molecular event that initiates the CRISPR interference, is carried out by the Cas5d protein in the subtype I-C organism Bacillus halodurans C-125. Such enzymatic activity has never been demonstrated for the Cas5 family of proteins, which are widely present in several CRISPR subtypes, and therefore warrants more careful investigation. Our results show that Cas5d recognizes both the base of the pre-crRNA stemloop and the 3′ single stranded (ss)-RNA sequence and cleaves the substrate in a metal-independent manner. The crystal structure of Cas5d reveals a ferredoxin-based architecture, with structural features and a cleavage site distinct from other known pre-crRNA processing factors. Mutagenesis indicates that residues Y46, K116, and H117 in Cas5d play critical roles in endoribonuclease activity, likely forming a catalytic triad. Furthermore, we show that after pre-crRNA cleavage, Cas5d assembles into a ∼400-kDa Cascade-like complex together with crRNA and the other two subtype-specific proteins Csd1 and Csd2, suggesting that Cas5d further participates in the crRNA-mediated DNA silencing step. This subtype I-C Cascade complex has much tighter affinity and greater specificity for the crRNA, a reflection of additional RNA contacts from other components of the Cascade complex. The B. halodurans strain C-125 encodes a subtype III-B (cmr1-6) and a subtype I-C CRISPR-Cas system. The latter system contains five CRISPR loci and seven cas genes (cas1-4, csd1-2, and cas5d) (Figure 1A ). The highly conserved repeats in the CRISPR loci encode a 7-bp stemloop flanked by the 2- and 11-nucleotide (nt) overhangs at the 5′ and 3′ ends, respectively. To identify the pre-crRNA processing factor in the subtype I-C CRISPR-Cas system, subtype-specific Cas proteins Csd1, Csd2, and Cas5d from B. halodurans C-125 were purified and incubated with a 32-nt RNA containing the conserved repeat sequence from B. halodurans C-125 CRISPR loci 3 (Figures 1B and 1C). Cas5d, but not Csd1 or Csd2, specifically cleaved the crRNA repeat at the 3′ base of the stemloop structure, between residues G21 and U22. crRNA repeat containing a 2′-deoxy substitution at G21 abolished Cas5d cleavage, confirming the cleavage site (data not shown). No further processing of the crRNA was observed when Csd1 and Csd2 were mixed with Cas5 (Figure 1C). We further verified that Cas5d was capable of processing a pre-crRNA containing multiple repeat-spacer-repeat sequences into the mature crRNAs (see Figure S1 available online). The endoribonucleolytic cleavage activity by Cas5d was metal independent, as addition of divalent cations (Mg2+, Mn2+, Ca2+, Ni2+, or Zn2+) or the metal chelator EDTA had no effect on cleavage rate and pattern (Figure 1D). Cas5d Cleavage produced a 2′,3′-cyclic phosphate that could be removed by T4 polynucleotide kinase (PNK) but not calf intestine phosphatase (CIP), and a 5′-OH detected by phosphorylation, in the 5′ and 3′ halves of the crRNA products, respectively (Figures 1E and 1F). This product feature and metal dependency study suggest that, similar to other characterized pre-crRNA processing factors, Cas5d most likely uses a general acid-base catalysis strategy to cleave the RNA. The substrate specificity of Cas5d was deduced by varying the pre-crRNA repeat sequence in a systematic fashion. The five CRISPR loci in B. halodurans C-125 converge into a highly conserved repeat sequence, with minor variations at the top of the stemloop and at the distal 3′ end (5′-GUCGCACUCUUCAUGGGUGCGUGGAUUGAAAU-3′; bold letters indicate variable sequence). This suggests that Cas5d likely processes all pre-crRNA transcripts from the five CRISPR loci (Figures 2A and 2B). A total of sixteen 5′ or 3′ fluorescently labeled RNA substrates were subjected to the cleavage assay by the Cas5d protein (Figures 2C and Table S1). Four titrations of Cas5d were used for each substrate to quantify the relative activity of Cas5d. Our results showed that recognition was weak to the distal end of the pre-crRNA stemloop, as replacing the UUCAU pentaloop with a stable GAAA tetraloop (RNA1 in Figure 2C). The base pairs at the base of the stemloop were systematically swapped (C3-G21, G4-C20, and C5-G19 to G3-C21, C4-G20, and G5-C19, respectively, in RNA 2, 3, and 4, Figure 2C), and a moderate inhibitory effect was observed as the swapping approached closer toward the cleavage site (RNA4 > RNA3 > RNA2). This suggests that there is stronger sequence-specific recognition toward the base of the stemloop. Interestingly, our data also revealed that, although deleting the 5′ GU overhang had little effect on Cas5d activity (RNA 6 in Figure 2C), there existed strong recognition to the 3′ overhang. Flipping it (from U22 to U32) to its complementary sequence completely abolished the Cas5d processing (RNA 5 in Figure 2C). Further deletion (RNA 7, 8, 9, 10) and point mutation (RNA 11–13) mapping revealed that the recognition focused mostly on the trinucleotide sequence U22G23G24 immediately following the cleavage site (Figure 2C). U22C, G23A, and G23U substitutions almost completely abolished Cas5d's activity (RNA 11, 12, and 14 in Figure 2C), and G24A and G24U had a moderate effect (RNA 13 and 15 in Figure 2C). These results are consistent with the CRISPR repeat conservation (Figure 2B). Taken together, Cas5d was able to efficiently process the minimum crRNA repeat substrate containing a 3-bp GAAA stemloop followed by a 3′ overhang as short as 3-nt (RNA 16 in Figure 2C). Because Cas5d is expected to tolerate the sequence variations among all B. halodurans CRISPR loci, we consider it to be the only pre-crRNA processor in B. halodurans. The strong recognition to the 3′ overhang region of the CRISPR repeats of Cas5d is similar to the behavior of subtype I-E Cse3 but different from that of subtype I-F Csy4. To gain deeper insight into the pre-crRNA cleavage mechanism, the crystal structure of the B. halodurans Cas5d protein was determined. Because the poor solubility of this protein hampered crystallization efforts, we purified Cas5d as an N-terminal SUMO fusion to high concentration and carried out in situ proteolysis in the crystallization drops by including the SUMO-protease at a 1:100 molar ratio. The resulting Cas5d crystals diffracted X-ray to 1.7 Å and allowed structure determination by single-wavelength anomalous dispersion (SAD) method from Se-methionine derivatized proteins (Figure 3A and Table 1). Two Cas5d molecules were observed in the asymmetric unit of the C2 space group (Figure S2); they agreed with each other with an rmsd of 0.23 Å for Cα atoms. Because Cas5d behaved as a monomer as judged by size-exclusion chromatography and dynamic light scattering measurement (data not shown), we hereby only describe the structure of one Cas5d molecule in the asymmetric unit. The electron density map of the Cas5d structure was well defined for most of the residues except the β7–β8 loop (Asn172-Leu182) and the C terminus (Val209-Glu236).Table 1Data Collection and Refinement Statistics of B. halodurans Cas5dCas5dNativeSe-MetData collection statisticsBeamlineCHESS A1CHESS A1Wavelength0.97700.9770Space groupC2C2Unit cell parameters (Å)a = 86.50, b = 46.68, and c = 129.03a = 86.61, b = 46.64 and c = 129.20β = 104.00°β = 104.70°Resolution (Å)20.0–1.70 (1.73–1.70)50.0–2.00 (2.03–2.00)Completeness (%)91.8 (76.7)96.6 (89.9)Redundancy3.1 (1.9)4.3 (2.7)I/σ(I)23.85 (2.79)20.40 (3.55)Rmerge (%)aRmerge = ∑h ∑i| I(h,ii) - < I (h) > | /∑h ∑iI (h,ii), where I (h,i) is the intensity of the ith measurement of reflection h and < I (h) is the mean value of I(h,i) for all i measurements.6.4 (20.5)11.6 (25.9)Refinement statisticsResolution (Å)20.0–1.70Rwork/Rfree(%)bRwork = ∑‖Fobs| - |Fcalc‖ / ∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree was calculated as Rwork using a randomly selected subset (5.1%) of unique reflections not used for structure refinement.15.65/22.01B-factor (Averaged, Å2) Protein24.18 Water35.61Rmsd Bond lengths (Å)0.019 Bond angles (°)1.685Ramachandran plot (%)cCategories were defined by Molprobity. Most favored100Highest-resolution shell is shown in parentheses.a Rmerge = ∑h ∑i| I(h,ii) - < I (h) > | /∑h ∑iI (h,ii), where I (h,i) is the intensity of the ith measurement of reflection h and < I (h) is the mean value of I(h,i) for all i measurements.b Rwork = ∑‖Fobs| - |Fcalc‖ / ∑|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree was calculated as Rwork using a randomly selected subset (5.1%) of unique reflections not used for structure refinement.c Categories were defined by Molprobity. Open table in a new tab Highest-resolution shell is shown in parentheses. Crystal structure of Cas5d revealed the presence of an N-terminal ferredoxin-like domain (Met1-Cys147) despite the lack of detectable homology at the primary sequence level. The topology of this domain (βαββββαβ) differs from other known ferredoxin folds (βαββαβ) by the insertion of a small lateral β sheet of β3–β4 (Figures 3A and S2). The α1 helix (Tyr35-Ile45) packs against the β sheet in the ferredoxin-like domain through extensive hydrophobic interactions. This helix connects to β1 and β2 through two flexible loops (Asp12-Tyr35 and Ile45-Thr50) (Figure S2). The α2 helix packs at the hydrophobic edge of β1 through hydrophobic interactions (Val5, Phe7, Ile45, and Val144 in ferredoxin fold; Leu121 and Leu125 in α2 helix) (Figure S2). A twisted β sheet (β7–β10, Lys160-Pro208) packs at the opposite edge of the ferredoxin-like domain. The continuous antiparellel β sheet in the ferredoxin domain generates a positively charged shallow groove of 13 × 15 Å2, forming a putative RNA binding pocket barricaded by the twisted β sheet and other upward flexible loops (see Discussion) (Figure 3B). Deletion of the twisted β sheet (Lys160-Pro208) significantly reduced but did not completely eliminate the endoribonuclease activity (Figure 3C, lane 4), suggesting that this region facilitates the binding of the pre-crRNA but probably does not contribute directly to the catalysis. The C terminus of Cas5d (Val209-Glu236), required for the solubility of the SUMO-Cas5d protein, adopts an extended conformation to contact the neighboring molecule (Figure S2). This unstructured loop is not conserved among the Cas5d proteins, and deletion of this region did not affect cleavage activity (Figure 3C, lane 3). The twisted β sheet (β7–β10) and the β3–β4 loop display more conformation dynamics, with significantly higher temperature B factors (∼58 Å2) than the core ferredoxin domain (∼21 Å2) (Figure S2). The shallow positively charged groove inside the ferredoxin domain of Cas5d is suggestive of the pre-crRNA binding site. Structure-guided mutagenesis was carried out to identify key RNA-binding and catalysis residues in Cas5d. Sequence alignment of the Cas5d family (sequence identity typically varied between 30% to 50%) from ten different organisms identified 23 highly conserved residues (Figure S3). An alanine scan was carried out for ten surface residues (E26, Y30, T34, S44, Y46, W47, K116, H117, R123, and R138) located in or near the putative pre-crRNA binding groove (Figure 4A ). Five of them (Y46A, W47A, K116A, H117A, and R123A) greatly reduced the cleavage activity of Cas5d (Figure 4C). Further quantification revealed that Y46A, W47A, and R123A mutants reduced the cleavage activity to ∼5%, ∼3%, and ∼10% of the wild-type level, respectively, and K116A and H117A mutations almost completely inactivated the enzyme (Figures 4D and 4E). These catalytically critical residues are clustered at a positive patch immediately outside the putative RNA-binding groove (Figure 4B). The close arrangement of Y46, K116, and H117, and the network of hydrogen bonds among them and D111, are suggestive of a catalytic triad that cleave the pre-crRNA through a general acid-base mechanism (Figure 4D). This is consistent with the metal-dependency study and the chemical structure of the cleavage products (Figure 1). Adjacent to the catalytic center, the vertically positioned W47 side chain stacks in front of the absolutely conserved P49 residue. We speculate that this residue stacks near the base of the pre-crRNA stemloop, serving to position the 3′ overhang near the catalytic center (see Discussion). The R123 residue in the α2-helix is separated from the Y46-K116A-H117 center by ∼9 Å and may be involved in recognizing the sugar-phosphate backbone or the nucleotide base directly. A series of experiments were carried out to investigate the function of the subtype I-C Cas5d protein after pre-crRNA processing reaction. Specifically, whether Cas5d, together with other subtype I-C-specific proteins Csd1 and Csd2, form in a multi-sub-unit protein complex as found in subtype I-E (Ecoli) and subtype I-F (Ypest) (denoted as Cascade in the subtype I-E) (Brouns et al., 2008Brouns 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: 960-964Crossref PubMed Scopus (1668) Google Scholar, Jore et al., 2011Jore M.M. Lundgren M. van Duijn E. Bultema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. et al.Structural basis for CRISPR RNA-guided DNA recognition by Cascade.Nat. Struct. Mol. Biol. 2011; 18: 529-536Crossref PubMed Scopus (420) Google Scholar, Wiedenheft et al., 2011Wiedenheft B. Lander G.C. Zhou K. Jore M.M. Brouns S.J. van der Oost J. Doudna J.A. Nogales E. Structures of the RNA-guided surveillance complex from a bacterial immune system.Nature. 2011; 477: 486-489Crossref PubMed Scopus (300) Google Scholar). Cas5d alone had rather weak affinity with the 5′ and 3′ halves of the pre-crRNA cleavage products as shown by the electrophoretic mobility shift assay (EMSA; Figure S4). This behavior is similar to that of Cas6 but differs from other processing factors, such as Cse3 and Csy4 (Gesner et al., 2011Gesner E.M. Schellenberg M.J. Garside E.L. George M.M. Macmillan A.M. Recognition and maturation of effector RNAs in a CRISPR interference pathway.Nat. Struct. Mol. Biol. 2011; 18: 688-692Crossref PubMed Scopus (134) Google Scholar, Jore et al., 2011Jore M.M. Lundgren M. van Duijn E. Bultema J.B. Westra E.R. Waghmare S.P. Wiedenheft B. Pul U. Wurm R. Wagner R. et al.Structural basis for CRISPR RNA-guided DNA recognition by Cascade.Nat. Struct. Mol. Biol. 2011; 18: 529-536Crossref PubMed Scopus (420) Google Scholar, Wang et al., 2011Wang R. Preamplume G. Terns M.P. Terns R.M. Li H. Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage.Structure. 2011; 19: 257-264Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Wiedenheft et al., 2011Wiedenheft B. Lander G.C. Zhou K. Jore M.M. Brouns S.J. van der Oost J. Doudna J.A. Nogales E. Structures of the RNA-guided surveillance complex from a bacterial immune system.Nature. 2011; 477: 486-489Crossref PubMed Scopus (300) Google Scholar). Mixing of B. halodurans crRNA, Cas5d, Csd1, and Csd2 proteins only led to small-sized and heterogeneous RNA-protein complexes as shown by size-exclusion chromatography (SEC; Figure S4). However, coexpression of CRISPR RNA, together with the Cas5d, Csd1, and Csd2 proteins in Escherichia coli BL21(DE3) cells, led to the formation of a stable ∼400-kDa complex as shown on SEC (F" @default.
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