CRISPR subtype I-A

SEE ALSO CRISPR-Cas

Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease Cas6^[1]

Clustered regularly interspaced short palindromic repeats (CRISPRs) confer adaptive immunity to prokaryotes through a small RNA-mediated mechanism. Specific endoribonucleases are required by all CRISPR-bearing organisms to process CRISPR RNAs into small RNA that serve as guides for defensive effector complexes. The molecular mechanism of how the endoribonucleases process the class of CRISPR RNA containing no predicted secondary structural features remains largely elusive. Here, we report cocrystal structures of a processing endoribonuclease bound with a noncleavable RNA substrate and its product-like fragment derived from a nonpalindramic repeat. The enzyme stabilizes a short RNA stem-loop structure near the cleavage site and cleaves the phosphodiester bond using an active site comprised of arginine and lysine residues. The distinct RNA binding and cleavage mechanisms underline the diversity in CRISPR RNA processing.

• (4ill).
• . Subunit A is colred in yellow and subunit B in cyan.

Cas6 proteins belong to the repeat-associated mysterious protein (RAMP) family of proteins that are characterized by, in most cases, a tandem ferredoxin fold. . SsCas6 comprises a tandem ferredoxin fold arranged side-byside and displays two distinct surfaces: one of β sheets and one of α helices. Like other RAMP proteins, both ferredoxin domains are interrupted by insertions to the canonical βαββαβ arrangement. The most notable is a three-helix insertion (α5–α7) following the first β strand (β6) of the C-terminal ferredoxin domain. Together with another helix insertion to the N-terminal ferredoxin fold (α2), these insertions form an important binding platform for the repeat RNA.

The overall structure of SsCas6-RNA is a 2:2 (protein/RNA) homodimer. There are two protein-RNA complexes in the asymmetric units of the 24-mer noncleavable complex crystals, whereas there are eight protein-RNA complexes in the 16-mer product-like complex crystal. However, the eight non-crystallographic symmetry-related protomers comprise only four pairs of the same homodimer as that of the 24-mer crystal. Although the isolated SsCas6 crystal has one protomer in its asymmetric unit, it again forms the same homodimer via crystallographic symmetry interactions. Thus, in all structures, SsCas6 or SsCas6-RNA complexes form the same homodimer mediated by protein residue interactions. The dimerization interface has a relatively extensive buried surface solvent accessible area (1,289 A˚²) that is suggestive of dimerization in solution and is made primarily of the helices (α5–α8) and β7 of its C-terminal ferredoxin domain. Cas6 dimerization has been previously observed in a noncatalytic Cas6 protein from Pyrococcus horikoshii (Ph) bound with its repeat RNA that, however, is mediated by the bound RNA molecules. The other three Cas6 do not seem to form dimers. Thus, dimerization is likely an inherent property of the protein unique to SsCas6. Structure superimposition revealed few differences in SsCas6 among the three structures with an exception for the β7–β8 hairpin loop. Structural differences among protomers or between the 24-mer and the 16-mer complexes are observed only in two termini of the bound RNA due to different packing interactions, suggesting that the observed core enzyme-RNA structure is independent of crystal-packing interactions.

The bound . Of the five loop nucleotides of the stemloop, three extrude out of helical stacking (A9, U11, and U13) and two stack on the three base-pair stem (C10 and A12). The whereas the single stranded regions have only limited contacts with the enzyme. SsCas6 residues that interact with RNA are labeled, colred in wheat, and shown in ball-and-stick representaion, RNA nucleotides that interact with the protein are colored magenta. The terminal nucleotides that do not contact SsCas6 (G1, C2, A21, A22, A23, and G24) are either disordered or are stabilized by crystal packing interactions. Thus the stem-loop is the primary structure recognized by SsCas6. . SsCas6 forms both nonspecific and specific interactions with the RNA. The most extensive interaction with the RNA phosphate sugar backbone is formed with the short stem, in particular, the 3' side of the stem (). The amino group of contacts the nonbridging oxygen atoms of both G15 and A16. The guanidinium group of of the G-loop further stabilizes the negative charge of nonbridging oxygen of G15. In addition, the side chain atoms of contact the nonbridging oxygen atoms of A14. In addition to electrostatic and hydrogen-bonding interactions with the phosphate backbone, SsCas6 forms base-specific interactions with the short stem. For the U6-A16 base pair, of the G-loop, respectively. Ser269 is well conserved whereas Tyr168 is often replaced by phenylalanine in SsCas6 homologs. . The . Arg268 of the G-loop lies near the Watson-Crick edge of G15 and forms base-specific interactions with the G15C7 base pair. . The fact that Arg268 is often replaced by lysine in other SsCas6 homolog suggests a possibility of coevolution between Cas6 and its substrates. The penta-nucleotide loop contacts SsCas6 through its two extruded nucleotides, U11 and U13, in a base-independent manner. . Although the exocyclic oxygen atoms of (in cyan), Asp48 is not well conserved, suggesting that the protein may be able to accommodate other nucleotides in the loop. The fact that there is no contact between the protein and the rest of the loop nucleotides suggests that both the size and the base identity of the loop are not critical to RNA recognition.

Examination of the SsCas6-24-mer and SsCas6-16-mer structures identified, surprisingly, four positively charged residues in its active site. These include Lys25 and Lys28 of α1 where the critical histidine of Cas6f and tyrosine of Cas6e are located, Lys51 on the loop connecting α2-β2, and Arg232 on the long β-hairpin loop where the critical serine of Cas6f residues. Observed separately in the 24-mer noncleavable and 16-mer product-like complex structures, the three lysine residues are within 3.5 A˚ to phosphate groups (Lys51 to A16, both Lys25 and Lys28 to A17) and Arg232 is close to 2'-OH of A16 (2.6 A˚). In the where the nucleophile 2'-OH group is missing, Arg232 is further away from the active site than that in the , suggesting its specific stabilization role for the 2'-OH group of A16.

Many CRISPR repeats contain no detectable palindromic feature, nor do they display conserved sequences. This raises the question of how CRISPR processing endonucleases recognize and cleave RNA transcribed from this group of CRISPR repeats. Both crystallographic and biochemical studies were shown that a CRISPR repeat RNA derived from a nonpalindromic repeat forms a short stem-loop motif, comprising minimally of two base pairs, near the site of cleavage. The endonuclease that cleaves this CRISPR repeat RNA, SsCas6, stabilizes and specifically recognizes the stem-loop motif. This mode of protein-RNA interaction results in a conformation of the scissile phosphate bond that enables its breakage. Mutations of SsCas6 residues that are predicted to stabilize this conformation are found to greatly reduce the RNA cleavage activity of SsCas6. The SsCas6-RNA complex structure suggests that extensive base pairing in RNA is not required for recognition by this class of processing endoribonucleases. The minimal two-base pair stem-loop formed near the cleavage site can be easily satisfied by many CRISPR repeat RNA devoid of stable secondary structures. Given the favorable conformation of the scissile phosphate bond for cleavage, we suggest that the observed SsCas6-RNA interactions near the cleavage site serve as a model for the class of endoribonucleases that recognize non-structured CRISPR RNA. Individual Cas6 proteins may further fine tune the specificity by recognizing base pairs and/or RNA nucleotides within and beyond the stem-loop motif. For instance, the previously studied PfCas6 endoribonuclease specifically recognizes the first eight nucleotides in addition to the cleavage site around nucleotide 22 (the wrap-around model). Although the structure of the RNA cleavage site bound to PfCas6 is not observed, it is tantalizing to imagine that this region also forms a short stem-loop. If so, a general recognition model of non-structured RNA may comprise both individually recognized peripherals and the cleavage site short stem-loop. Significantly, the short stem-loop resembles the structure at the cleavage site of the CRISPR RNA derived from palindromic repeats. This common motif near the RNA cleavage site establishes a mechanistic link in the catalytic process of the endoribonucleases that process two different types of RNA. The discovery of an arginine residue in SsCas6 at the position typically occupied by the general base in metal-independent endoribonucleases is surprising. Although arginine has a favorable electrostatic property for stabilizing the developing charge in the pentavalent transition state during phosphodiester bond cleavage reaction, the naturally high pKa of its guanidinium group argues against its role as a general base. However, the functional groups tentatively assigned as the general base in several ribozymes also have naturally high pKa. In these cases, it has been proposed that active site environment may lower the pKa values of these groups. Whether this is the case for the arginine of SsCas6 awaits additional careful dissection of the catalytic mechanism.

Additional representatives:

• PhoCas6nc (Cas6a) from Pyrococcus horikoshii (3qjl). . Other representatives: 3qjj, 3qjp. ^[2]
• SsoCas6-1B (Sso1437, Cas6-1 family, SsoCas6) from Sulfolobus solfataricus (3zfv). . ^[3].