Structural basis for assembly of non-canonical small subunits into type I-C Cascade

Abstract

Bacteria and archaea employ CRISPR (clustered, regularly, interspaced, short palindromic repeats)-Cas (CRISPR-associated) systems as a type of adaptive immunity to target and degrade foreign nucleic acids. While a myriad of CRISPR-Cas systems have been identified to date, type I-C is one of the most commonly found subtypes in nature. Interestingly, the type I-C system employs a minimal Cascade effector complex, which encodes only three unique subunits in its operon. Here, we present a 3.1 Å resolution cryo-EM structure of the Desulfovibrio vulgaris type I-C Cascade, revealing the molecular mechanisms that underlie RNA-directed complex assembly. We demonstrate how this minimal Cascade utilizes previously overlooked, non-canonical small subunits to stabilize R-loop formation. Furthermore, we describe putative PAM and Cas3 binding sites. These findings provide the structural basis for harnessing the type I-C Cascade as a genome-engineering tool.

Structural and biochemical insights into CRISPR RNA processing by the Cas5c ribonuclease SMU1763 from Streptococcus mutans

“…This is consistent with the prediction of a dimeric biological unit using the PDBePISA tool (44) formed mainly by interactions between the N-terminal strands (Asn2-Arg5) and β4-α2 loops of two protomers with buried surface area 950 Å 2 (compared to 2,300 Å 2 for the DvuCas5c/Cas7 interface area in the D. vulgaris Cascade complex) (Fig. 3B) (42). The PDBePISA analysis also predicted a dimeric state for BhaCas5c, which was verified using size-exclusion chromatography using the purified BhaCas5c.…”

“…These results indicate that SmuCas5c exhibits a greater tolerance to substrate sequence variations compared to BhaCas5c including mutations located downstream of the cleavage site. Cascade-crRNA complex, the DvuCas5c Trp51 is involved in crRNA binding with the side chain positioned between the first two bases (U1 and G2) of the crRNA 5´-handle (42). In the SmuCas5c active site, the smaller side chain of His51 can also interact with crRNA bases, but it has different steric properties compared to Trp regarding to substrate coordination (Fig.…”

“…Cas5c and Cas6 enzymes have been proposed to use a general acid-base mechanism based on a catalytic triad of His, Tyr, and Lys, which may function as a general base, a general acid, and in intermediate stabilization, respectively (22,23,32,49). Previous studies on DvuCas5c suggested that the catalytic triad of this enzyme includes Tyr46, Lys116, and His117, which are located close to the 3´-end of crRNA in the D. vulgaris Cascade-crRNA complex (32,42). Metal ionindependent RNase activity and RNA cleavage products of SmuCas5c also suggest that this enzyme cleaves crRNA substrates using a general acid-base mechanism similar to that of RNase A and RNA splicing endonucleases (51,52).…”

Espectrometria de Massa na Análise de Sistemas Biológicos

“…A IST permite a fragmentação dos complexos proteicos na frente do espectrómetro de massa a jusante da fonte de ativação (MS/MS), o que permite fragmentar os monómeros libertados com CID ou UVPD (MS3) [68,72,101]. Recentemente a IST foi utilizada para caracterizar sistemas CRISPR [102]. A dissociação do complexo com IST permitiu validar a informação obtida por Cryo-EM e identificar os monómeros que compõem o complexo e a sua massa, como demonstrado na Figura 6.…”

“…As cores na estrutura representam: Cas7, a azul e cinzento; Cas8c, a roxo; Cas5, a laranja; Cas11c, a amarelo e vermelho; crRNA ligado às cinco proteínas, a verde. Adaptada com permissão da referência [102].…”