Recognition of a pseudo-symmetric RNA tetranucleotide by Csx3, a new member of the CRISPR associated Rossmann fold superfamily

Abstract

The CRISPR/Cas adaptive immune system shows extreme diversity in the number of CRISPR/Cas types and subtypes, and in the multitude of CRISPR associated protein families of which they are composed. Despite this diversity, the roles of many Cas protein families are now defined with regard to spacer acquisition, crRNA biogenesis, and DNA or RNA surveillance and targeting. However, a number of unclassified CRISPR-Cas proteins remain. Such proteins have traditionally been designated as CRISPR subtype x (Csx). Here we revisit the structural analysis of one such protein, Csx3, and show that this homodimeric protein utilizes a Rossmann fold for the recognition of an RNA tetranucleotide. Tertiary and quaternary structural similarities of Csx3 to CRISPR/Cas proteins Csx1 and Csa3 are identified and suggest Csx3 is a new member of the CRISPR Associated Rossmann Fold (CARF) superfamily. The structure of the Csx3/RNA complex illustrates one way CARF domain proteins may recognize pseudo-symmetric polynucleotides.

Keywords: CARF; CRISPR; CRISPR/Cas; Cas; Csa3; Csm6; Csx1; Csx3.

Figure 1.

RNA recognition by AfCsx3. A) Stereo ribbon diagram of AfCsx3 Chain A bound to a ssRNA (PDB ID: 3WZI). Beta strands, α helices and the conserved β3-α2 loop (Ser44-Ile49) are shown in green, yellow and magenta, respectively. Secondary structures are labeled in order of their occurrence in the amino acid sequence. The orientation is roughly equivalent to that of Fig. 1 in Yan et al., who modeled nucleotides N2 and N3 into subunit A which are shown as “sticks” and labeled accordingly. B) A topology or fold diagram of Csx3. Beta strands and α helices are colored according to panel A. Gradients are added to show the N-to-C directionality of the individual secondary structures, morphing from dark to lighter colors. The boxed region indicates the Rossmann fold present in Csx3. C) The similarly depicted RRM or ferredoxin-like fold. This fold is substantially different from the Csx3 fold in panels A and B, where Csx3 lacks the 4-stranded antiparallel β-sheet and the antiparallel helices of the RRM fold. D) Fold of the classic dinucleotide binding domain composed of 2 successive Rossmann folds. The first Rossmann fold is contained within the boxed region and is aligned with the Rossmann fold present in Csx3 in order to highlight the similarities between the 2 folds. Helix α3 of Csx3 runs along the back side of the mixed β sheet, contacting strands β4, β3 and β2, but has been slightly repositioned in panel B in order to accentuate the similarities between the Rossmann folds present in the classic dinucleotide binding domain and that in Csx3.