Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

Figure 1.

S. epidermidis RP62a CRISPR–Cas locus and effector complex schematics. (A) Schematic of S. epidermidis RP62a CRISPR–Cas locus architecture. The locus is composed of the leader (gray rectangle) and tandem copies of the repeats (black diamonds) and spacers (numbered 1–3), followed by the nine associated protein-coding cas genes. Five proteins (Cas10, Csm2–5) associate to form the effector complex. (B) Sequences of the three crRNAs associated with the S. epidermidis RP62a repeat-spacer array shown in A. Repeat sequences are shown in black and spacer sequences for crRNA 1–3 are shown in green, orange, and magenta, respectively. Intermediate and mature lengths are denoted by a dotted line with the respective length listed above. (C) Schematic diagram of a proposed model of the Type III-A CRISPR effector complex with a 43 nucleotide (nt) CRISPR RNA (crRNA). The effector complex model is composed of five copies of Csm3 (yellow), three copies of Csm2 (orange), and one copy each of Csm4 (green), Csm5 (teal) and Cas10 (red), in addition to the 43 nt crRNA (black line) (28).

Figure 2.

Molecular architecture of S. epidermidis Csm2. (A) Two perpendicular views of the crystal structure of SeCsm2 show an exclusively α-helical fold consisting of 6 helices. SeCsm2 is colored in a rainbow from the N- (blue) to C- (red) termini. A portion of the N-terminal affinity tag was present in the density and is included in the structure. (B) Structural alignment of SeCsm2 (green) and the putative monomeric version of TmCsm2 (76) (magenta). The overall architecture is similar with a root-means-square-deviation (RMSD) between structures of 1.98 Å. One significant difference is the rearrangement of TmCsm2 α₃ to form two helices (α₄–α₅) in SeCsm2 (dashed oval). (C) Structural alignment of SeCsm2 α₄–α₅ (green) with Type III-A TmCsm2 α₃ (magenta) and Type III-B TtCmr5 α₄–α₅ (78) (blue). The helix architecture of Type III-A and Type III-B Csm2 homologs reveals an intermediate organization in SeCsm2. (D) SeCsm2 accessible surface colored by electrostatic potential calculated with APBS (72). Two small positive regions (blue) are found on opposite sides of the molecule. One negative region (red) is shown at the opposite end from the positive regions. The bar at the bottom maps the electrostatic potential values from –10 k_BT/e_c [red] to 10 k_BT/e_c [blue].

Figure 3.

Molecular architecture of S. epidermidis Csm3. (A) Two perpendicular views of the crystal structure of SeCsm3 show that it is composed of an RRM fold consisting of a five-stranded anti-parallel β-sheet and four helices, a β-hairpin, and an isolated α-helix. Sheets are colored in red and helices in blue. (B) Topology diagram of SeCsm3 shows that the N- and C-terminal sequences form the RRM fold (dashed oval), with the other domains located at the center of the polypeptide. (C-E) Structural alignment of Cas7 family proteins. SeCsm3 is aligned with (C) MjCsm3 (82) (RMSD of 1.68 Å over 134 C_α atoms), (D) MkCsm3 (86) (RMSD of 1.98 Å over 153 C_α atoms), and (E) SsoCas7b (88) (RMSD of 2.99 Å over 122 C_α atoms). The β-hairpin (dashed oval) and helix (dashed rectangle) in SsoCas7b are rotated with respect to the ones in SeCsm3. The alignments show the wide variation in backbone subunit architecture of effector complexes of different types and subtypes.

Figure 4.

Purification and characterization of the S. epidermidis CRISPR effector complex. (A) Coomassie blue stained 12% SDS-PAGE of the purified SeCas10–Csm effector complex. The gel shows fractions collected around the peak eluted from the column. All five proteins expected in the complex are present in the peak. The identity of the proteins is shown on the right. The fraction enclosed by the dashed rectangle was used for EM structural studies. M, molecular weight markers. (B) Superdex 200 trace of the SeCas10–Csm effector complex purification. Indicated peaks correspond to the column void volume (I), SeCas10–Csm aggregate peak (II), SeCas10–Csm non-aggregate peak (III), and contaminants (IV). The horizontal red line marks the elution fractions loaded into the gel shown in A. (C) Mass spectrometry of crRNA isolated by phenol-chloroform extraction from the purified complex used for EM studies. The two crRNA species were determined to be a 37-nucleotide mature crRNA with the sequence from Spacer 1 (green sequence) and a 43-nucleotide mature crRNA with the sequence from Spacer 2 (orange sequence). The corresponding molecular weight and sequence are shown for each peak of interest. (D) Time course target RNA cleavage assay of the purified SeCas10–Csm effector complex. Cleavage was monitored using a 5′-FAM-labeled target RNA (blue sequence) complementary to the 37 nt Spacer 1 sequence (green sequence). Cleavage activity was assessed in the presence and absence of magnesium. Reactions were run on a 15% Urea-PAGE in order to visualize cleavage activity.

Figure 5.

Overview of the cryo-EM structure of the major filament subcomplex. (A) A 5.2 Å cryo-EM reconstruction of the SeCas10–Csm subcomplex shows a helical stem region adjoining the non-helical base region. The stem is formed by five SeCsm3 subunits while the base includes SeCsm4 and SeCas10. (B) SeCsm3 and a putative SeCsm4 RRM are docked into the cryo-EM density map. Five SeCsm3 (alternately colored orange and yellow) and one SeCsm4 RRM (blue) were confidently placed in the density. Crystal structures of homologous structures or homology models for SeCas10 and SeCsm4 did not fit well into the density at the base. Additional density at the base was putatively assigned as SeCas10 (dashed oval). The SeCsm3 crystal structures and the SeCsm4 RRM form a helical arrangement, with the RRM β-strands facing the outside of the helix and the β-hairpin on the inside of the helix. (C) Difference density map between the experimental map and the model of the subcomplex reveals additional density along the SeCsm3 stem, which corresponds to the β-hairpin in SeCsm3 not present in the crystal structure as well as the crRNA traversing the length of the stem. Regions in the dashed box are shown in D and E. (D) Close-up views of the SeCsm3 secondary structure elements show good agreement with the cryo-EM density. (E) Difference density map shows that disordered regions in the SeCsm3 crystal structure have clear density in the cryo-EM map. These regions of density were putatively assigned as an extension of each SeCsm3 β-hairpin (dashed lines).

Figure 6.

The effector subcomplex shows the crRNA in a conserved, positively charged channel. (A) Two orthogonal views of the cryo-EM map colored by subunit identity. Cryo-EM density was assigned to each subunit in the complex after crystal structure docking. Density is assigned for five SeCsm3 (gold and yellow), one SeCsm4 (blue), and one SeCas10 (green), with additional density within each SeCsm3 putatively assigned as the crRNA (purple). (B) Putative crRNA density isolated from the SeCas10–Csm subcomplex shown in A. The density follows a continuous path along the length of the stem. (C) The SeCsm3 stem is shown in accessible surface representation colored by electrostatic potential calculated using APBS (72). A highly positively charged channel (blue) is seen traversing the length of the stem. (D) Putative crRNA density (purple) overlaid on the electrostatic potential map of SeCsm3 stem. The positively charged channel surrounds the negatively charged crRNA, providing further evidence that the density corresponds to the crRNA. The bars at the bottom of C and D map the electrostatic potential values from –6 k_BT/e_c [red] to 6 k_BT/e_c [blue]. (E) Schematic diagram showing the composition of the SeCas10–Csm subcomplex containing a 43 nt crRNA. Csm2 and Csm5 are not present in the subcomplex.