Structure and RNA-binding properties of the type III-A CRISPR-associated protein Csm3 - PubMed (original) (raw)

. 2013 Nov;10(11):1670-8.

doi: 10.4161/rna.26500. Epub 2013 Sep 30.

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Structure and RNA-binding properties of the type III-A CRISPR-associated protein Csm3

Ajla Hrle et al. RNA Biol. 2013 Nov.

Abstract

The prokaryotic adaptive immune system is based on the incorporation of genome fragments of invading viral genetic elements into clusters of regulatory interspaced short palindromic repeats (CRISPRs). The CRISPR loci are transcribed and processed into crRNAs, which are then used to target the invading nucleic acid for degradation. The large family of CRISPR-associated (Cas) proteins mediates this interference response. We have characterized Methanopyrus kandleri Csm3, a protein of the type III-A CRISPR-Cas complex. The 2.4 Å resolution crystal structure shows an elaborate four-domain fold organized around a core RRM-like domain. The overall architecture highlights the structural homology to Cas7, the Cas protein that forms the backbone of type I interference complexes. Csm3 binds unstructured RNAs in a sequence non-specific manner, suggesting that it interacts with the variable spacer sequence of the crRNA. The structural and biochemical data provide insights into the similarities and differences in this group of Cas proteins.

Keywords: Cas7; RAMP; RRM domain; adaptive immunity; ferredoxin domain.

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Figures

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Figure 1. Structure of Methanopyrus kandleri Csm3. (A) The structure of Mk Csm3 can be divided into four distinct elements: the core (green) and lid domain (blue), a helical N-terminal (red), and a C-terminal domain (yellow). The structural elements of the core adopt a ferredoxin-like fold with β-α-β-β-α-β arrangement. The core is topologically interrupted by multiple insertions forming the lid and the helical N-terminal domain. The C-terminal domain packs against the core and is of mixed structural composition. The dashed blue line represents the missing disordered region between residues 200 and 214. The two views are related by a 180° rotation as indicated. (B) Topology diagram of Mk Csm3. Helices are represented as circles and β-strands as arrows. The secondary structure elements have been labeled numerically maintaining the nomenclature of RRM domains. The β-strands of the C-terminal domain extending the RRM β-sheet have also been labeled numerically. The additional α-helices have been labeled with letters (αA to αL). (C) A structural zinc ion present in the helical N-terminal domain is shown as a gray sphere, together with the coordinating residues (a cysteine and three histidine residues).

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Figure 2. Structural similarity between Csm3 and Cas7. (A) Sso Cas7 (PDB ID: 3PS0, rmsd: 4.2Å, blue) shares the highest structural homology with Mk Csm3 (gold) beyond the core domain (gray). Both proteins have a similar arrangement of auxiliary domains surrounding the RRM-like fold, as well as a conserved architecture of the C-terminal domain. (B) Topology diagram of Mk Csm3 and Sso Cas7 showing the connectivity of the RRM fold relative to the other domains. The topological arrangement of the insertions is similar in both proteins. Similarities in secondary structure elements are highest within the core and low in the auxiliary domains.

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Figure 3. RNA-binding properties of Csm3. (A) Mk Csm3 binds to a physiological crRNA substrate (left panel). 32P-labeled crRNA transcripts were incubated in the absence or presence of 5 µM, 10 µM, and 20 µM Mk Csm3. (B) Electrophoretic mobility shift assays were performed with the respective [32P]-5′-end labeled RNAs and increasing concentrations of Mk Csm3 (0 µM, 1 µM, 30 µM, 100 µM). Mk Csm3 binds to single-stranded RNA substrates (lane 16–20) but not significantly to the repeat sequences (lanes 1–5 and 6–10). Binding to single-stranded RNA is dependent on length but not sequence (compare lanes 16–20 and 11–15). Weak binding of Mk Csm3 to processed and unprocessed repeat sequences (lanes 1–5 and 6–10, respectively) is likely attributed to the ssRNA overhangs. (C) Methanopyrus kandleri repeat sequence conservation and predicted RNA folding.

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Figure 4. Identification of Csm3 RNA-binding residues. (A) The structure of Csm3 is shown in surface representations, in the same orientations as in Figure 1A, colored according to electrostatic potential. Charged patches (blue) are present at the back of the lid domain as well as at the interface between the core and N-terminal helical domain. Negatively charged surfaces (red) are located along the front of the N-terminal insertion and cover the C-terminal domain. Two surface patches discussed in the text (patch 1 and 2) are indicated. (B) Corresponding surface representations of Csm3 colored according to conservation with the Csm3 family. The conservation is based on a comprehensive alignment (Fig. S4B). Increase in conservation is shown in increasingly darker shades (from to red). No or low conservation (white and yellow) is found in the N-terminal insertion and the C-terminal domain. Highly conserved residues (orange and red) are located within the lid (patch 1) and core domains (patch 2) and coincide with positively charged surfaces (A). (C) Sequence alignments of Csm3 orthologs in regions corresponding to surface patches 1 and 2 (A and B). Residues selected for mutation analysis are highlighted with red dots. The unstructured loop (H199-S214) replaced by a (GS)3 linker is represented as a dashed red line. (D) RNA binding of Csm3 mutants to a single-stranded RNA substrate U15. Wild-type (WT) protein and the double mutation within the core domain (patch2) bind with comparable affinity. Replacement of the unstructured loop (H199-S214) by a -(GS)3- linker does not impair binding, while the single mutation R21A has completely lost RNA binding ability at this condition. (E) Coomassie-stained 12% SDS-PAGE gel of the purified protein samples used in the assays.

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