CRISPR subtype I-B

SEE ALSO CRISPR-Cas

Crystal structure of CRISPR RNA processing endoribonuclease Cas6b^[1]

A subclass of recently discovered CRISPR repeat RNA in bacteria contains minimally recognizable structural features that facilitate an unknown mechanism of recognition and processing by the Cas6 family of endoribonucleases. Cocrystal structures of Cas6 from Methanococcus maripaludis (MmCas6b) bound with its repeat RNA revealed a dual site binding structure and a cleavage site conformation poised for phosphodiester bond breakage. Two non-interacting MmCas6b bind to two separate AAYAA motifs within the same repeat, one distal and one adjacent to the cleavage site. This bound structure potentially competes with a stable but non-productive RNA structure. At the cleavage site, MmCas6b supplies a base pair mimic to stabilize a short 2 base pair stem immediately upstream of the scissile phosphate. Complementary biochemical analyses support the dual-AAYAA binding model and a critical role of the protein-RNA base pair mimic. Our results reveal a previously unknown method of processing non-stem-loop CRISPR RNA by Cas6.

Methanococcus maripaludis C5 (Mm) contains a type I-B Cas6, MmCas6b (MmarC5_0767), responsible for processing crRNA. The RNA substrate of MmCas6b, the NC_009135 repeat, contains 37 nucleotides. To understand the biochemical mechanism of Cas6b, two cocrystal structures of MmCas6b bound with its RNA substrate analogs were determined. The complex structure with a at 3.0 A˚ resolution reveals two unexpected dual binding motifs optimal for cleavage site recognition (4z7k). The complex structure with a at 3.5 A˚ resolution (4z7l) reveals negligible structural changes in the active site despite the lower stability of the complex, suggesting a role of the distal minor motif in aiding substrate recognition rather than catalysis. Dual recognition by Cas6 reconciles the previous inconsistent binding data among Cas6 proteins and provides a strategy for Cas6 endoribonucleases to process CRISPR repeats lacking stable stem-loop structures.

Although free energy minimization predicts a stem-loop structure for the NC_009135 repeat RNA in isolation, it forms two recognition motifs (I and II) when bound to MmCas6b. comprising nucleotides 16–30 including the cleavage site between nucleotides 29 and 30 (motif II). The two motifs are linked by three extended nucleotides 13–15 (40 A˚), which results in few contacts between the two bound MmCas6b subunits with only 235 A˚² buried solvent-accessible surface area.

To assess the impact of the distal motif I on the structure of the MmCas6b-RNA complex, a crystal structure of MmCas6b bound with a nucleotides excluding the cleavage site (nucleotides 16–29) was also analyzed. (4z7l). The homodimer buries a 598 A˚² solvent-accessible area averaged among the three complexes in the asymmetric unit, suggesting a weak but discernible dimerization interaction.

Motif II of the repeat RNA is the major site recognized by MmCas6b. It comprises a 2 base pair stem (G16-C29/C17-G28) and an adenine-rich loop (A18-A27). The major groove of the short stem faces the interior of MmCas6b, and together with the . Other residues do not form base-specific hydrogen bonds but pack around the two base pairs. These include Gln183 of loop β6-β7, Asn39, His40, and of loop α1-β2, and Asn207 and Ser209 of the G loop. The adenine-rich loop wraps mostly around the α3 helix and partially around the αA helix and contacts several asparagine residues via its curved phosphosugar backbone. Only A26 and A27 of the loop are involved in base interactions with the main chain atoms of the MmCas6b residues. and its A₈AUAA₁₂ loop is nearly identical to Motif II A₂₃ACAA₂₇ . Consequently, the two motifs share the AAYAA (Y=C, U) sequence and use the same surface of the subunit B to interact with the adenine-rich loop. . The most unusual feature of MmCas6b-RNA interaction involves a protein-RNA base pair mimic between Tyr47 and A18. The . The Tyr47-A18 base pair mimicry is enabled by unstacking and splaying of the A-rich loop that gives away space for Tyr47 to stack on top of the 2 base pair stem. Arg206 forms hydrogen bond with the G16-C29 pair. In addition, . This mode of .

• .
• A₂₃ACAA₂₇.

Phosphodiester bond cleavage by MmCas6b takes place . The nucleotide A30 splays away from the helical track of the stem toward the α1 helix. The rotation of A30 around the scissile phosphate bond is critical to formation of the conformation favorable for catalysis. Due to the use of 2'-deoxy modification of C29 in crystallization, the 2'-hydroxyl oxygen is absent from our structure. However, if it were present, the three reactive atoms would form the inline conformation. . These residues are colored in violet. is in a position to donate proton to the leaving 5'-oxygen. probably stabilizes the negative charge of the transition state. The imidazole ring of His38 interacts with the splayed A30 nucleotide. Consistently, mutations of His40 and His38 both reduced the cleavage activity, and their double mutation abolished cleavage. Interestingly, mutation of Arg24 or Lys29 to alanine did not reduce RNA cleavage activity, suggesting the redundant roles of these residues in RNA cleavage. Alternatively, MmCas6b relies primarily on the general base His40 and less so on a general acid for protonating the leaving oxygen. .

The X-ray crystal structure of a Cas6 crRNA processing endonuclease bound with its RNA substrate analogs was determined. MmCas6b cleaves within the repeat region of the precursor crRNA 8 nt from its 3' end. The structure of MmCas6bRNA complex revealed a and a second MmCas6b subunit bound to a minor recognition site. This secondary structure permits proper recognition of the cleavage site in spite of a competing, potentially more stable, structure. The 2 base pair stem immediately upstream of the cleavage site facilitates the inline conformation of the scissile phosphate bond required for cleavage. This mode of Cas6-RNA recognition illustrates the power of protein enzymes in shaping structurally flexible RNA for catalysis.

MmCas6b is a member of Cas6 RNA processing endoribonucleases found in bacteria and archaea whose RNA substrates have a wide range of structural features. While recognition and processing of stable stem-loop RNA are well understood due to several high-resolution structures of Cas6 bound with their respective stem-loop RNA substrates, these processes are not clear for repeat RNA lacking stable stem loops. One method of processing is for Cas6 to impose a stem-loop structure of the RNA that is otherwise unstable in isolation. Sulfolobus solfataricus Cas6 bound with its short repeat RNA (24mer) illustrates this principle. However, the extent to which Cas6 stabilizes a short stem-loop structure is limited for long repeat RNA containing potentially competing secondary structures. MmCas6b structures reported here provide a second method of recognition in which the free 5' end of the repeat is stabilized by one Cas6 subunit so the . Although in this case the 5' distal recognition was shown to be non-essential to cleavage, other Cas6 may have evolved to depend more critically on sites beyond the cleavage site to ensure a catalytically competent conformation at the cleavage site. The added binding site(s) for Cas6 may have an evolutionary advantage in mesophilic organisms where it is difficult to dissolve competing structures in long pre-crRNA transcripts. Pyrococcus furiosus Cas6 may also adopt this method of recognition with a stringent requirement for the presence of the 5' terminus of the repeat RNA. Consistently, crystal structures of PfCas6 and its close homolog Pyrococcus horikoshii Cas6 reveal their specific interactions with the 5' terminus of the repeat RNA. The dual binding model allows Cas6 to overcome the challenge presented in the repeat RNA containing competing and non-productive secondary structures and may thus have general applicability in more Cas6 processing systems.

CRISPR subtype I-B?

• TthCas6B (TTHB231) from Thermus thermophilus. (4c9d). ^[2]. Other representative: 4c98.