Structures of an active type III-A CRISPR effector complex

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.

Keywords: CRISPR; complex with ATP; complex with target RNA; crRNA; cryo-EM; structure; type III-A.

Figure 1.. Schematic diagram of the S. epidermidis RP62a CRISPR-Cas locus and purification of the S. epidermidis effector complex.

(A) The S. epidermidis CRISPR-Cas locus organization. The leader sequence (light gray rectangle) is followed by spacer sequences (1, 2, 3) interspersed with tandem repeats (black squares). There are nine protein coding cas genes, including five (Cas10, Csm2-5) that associate to form the CRISPR-Cas effector complex. (B) Schematic diagram of the effector complex bound to a 37 nt crRNA. Four subunits of Csm3 (light green) and three subunits of Csm2 (light blue) compose the helical backbone, while one subunit of Csm5 (purple) or Csm4 (dark green) and Cas10 (dark blue) cap the ends. A 37 nt crRNA (black) is illustrated to show the arrangement of nucleotides when bound to the effector complex. (C) The three crRNA sequences found at the S. epidermidis RP62a CRISPR locus. Repeat sequences (black) flank the cRNA spacer sequences shown in green, orange, and yellow. Intermediate (71 nt) and mature (37 – 43 nt) crRNA lengths are marked by a dotted black line. D) Purification of the S. epidermidis effector complex. Representative Coomassie stained SDS-PAGE gel that shows S. epidermidis fractions eluted from a Superdex 200 column (GE). A molecular weight marker ladder (PM) is shown on the left and the complex subunits are labeled on the right. (E) A representative chromatogram resulting from passing effector complex over a Superdex 200 column. The peaks are identified as the column void volume (I), aggregated peak (II), and non-aggregated peak (III). The fractions from peak III are shown in the gel in panel D. (F) A 15% Urea-PAGE gel showing the cleavage of two different target RNA oligomers at different time points. Cleavage was monitored using a 5’-FAM labeled target RNA. The sequences of the RNA oligomers used in this experiment are detailed in the Key Resources Table. These lanes are found flanking the Target 1 reaction lanes in the middle and show no cleavage. The gel was visualized on a Sapphire Imager (Azure Biosystems).

Figure 2.. The apo effector complex maps.

(A) A 3.55 Å map of the S. epidermidis effector subcomplex shown in two different orientations. (B) Atomic model of the effector subcomplex placed into the reconstructed volume. (C) A 4.45 Å map of the apo S. epidermidis effector complex shown in two different orientations. (D) Atomic model of the tall apo effector complex placed into the reconstructed volume. (E) A 4.97 Å map of the short apo S. epidermidis effector complex shown in two different orientations. (F) Atomic model of the short apo effector complex placed into the reconstructed volume. For all panels, in the cryoEM maps (left and central columns) the region corresponding to Cas10 is shown in green, to Csm4 in salmon, to the Csm3 subunits in shades of blue and cyan, to the Csm2 subunits in shades of yellow, to Csm5 in purple, and to the crRNA in orange. In the atomic models, the three Csm3 subunits are shown in different shades of blue and cyan, Csm4 is shown in salmon, the two Csm2 subunits are shown in shades of yellow, Csm5 is shown in purple, Cas10 is shown in green, and the crRNA is shown in orange with the bases shown in stick representation. The cryoEM maps on the right column are shown in gray at a 5 σ contour level.

Figure 3.. Cryo-EM structures of the effector complex in the ATP and target RNA bound states.

(A) A 3.87 Å map of the tall ATP bound S. epidermidis effector complex shown in two different orientations. (B) Atomic model of the tall ATP bound effector complex placed into the reconstructed volume. (C) A 3.67 Å map of the short ATP and self target RNA bound S. epidermidis effector complex shown in two different orientations. (D) Atomic model of the short ATP and self target RNA bound effector complex placed into the reconstructed volume. (E) A 4.06 Å map of the non-self target RNA bound S. epidermidis effector complex shown in two different orientations. (F) Atomic model of the non-self target RNA effector complex placed into the reconstructed volume. For all panels, in the cryoEM maps (left and central columns) the region corresponding to Cas10 is shown in green, to Csm4 in salmon, to the Csm3 subunits in shades of blue and cyan, to the Csm2 subunits in shades of yellow, to Csm5 in purple, and to the crRNA in orange. In the atomic models, the three Csm3 subunits are shown in different shades of blue and cyan, Csm4 is shown in salmon, the two Csm2 subunits are shown in shades of yellow, Csm5 is shown in purple, Cas10 is shown in green, and the crRNA is shown in orange with the bases shown in stick representation. The cryoEM maps on the right column are shown in gray at a 5 σ contour level.

Figure 4.. Repeated protein interactions form the helical structure of the effector complex.

(A) A zoomed in view of the thumb of Csm4 (red) holding the crRNA against the fingers, and palm of Csm3.1 (slate blue). (B) A zoomed in view of the interaction between Csm5, shown in purple, and Csm3.4, shown in deep teal. (C) A magnified view of the interface that Csm2.1 (pale yellow) forms with Cas10 (green) and Csm 2.2 (yellow). (D) Magnified view of the interaction between Csm2.3, shown in wheat, and Csm5, shown in purple.

Figure 5.. The crRNA forms a repeating pattern as it travels up the effector complex.

(A) A magnified view of the 5’ target sequence, specifically A (−8) of the crRNA interacting with F40 and H291 of Csm4. F40 and H291 are shown in red as sticks. (B) A view of the 5’ target sequence as it travels up through Csm4. F249 of Csm4 stacks with A (−5) and Y148 stacks with A (−2). F249 and Y148 are depicted in red as sticks. (C) A view of the 6 nucleotide repeating pattern of the crRNA as it travels through Csm3.3 and 3.4. (D) A magnified view of the thumb of Csm3.2 holding the crRNA agains the fingers and palm of Csm3.3. (E) A magnified view of the Csm3.3 thumb holding the crRNA against the fingers and palm of Csm5. (F) The tall apo effector complex shown as an electrostatic potential surface. Across from each Csm3 thumb is a basic patch on Csm2.

Figure 6.. The self target RNA and non-self target RNA bound effector complexes highlight how the S. epidermidis effector complex binds to target RNA and ATP.

(A) A magnified view of the short ATP and self target RNA bound effector complex superposed with the short apo effector complex Cas10 (shown in gray). The Cas10 from the ATP and self target RNA bound effector complex is shown in green with the newly structured loop depicted in yellow. The crRNA is shown in orange with the bases depicted as sticks in cyan, and that target RNA is shown in orange with the bases depicted as sticks in violet. (B) Superposition of the short apo complex (shown in gray) and the short ATP and self target RNA bound effector complex (subunits coloring shown in figure). (C) A magnified view of the Csm3.3 (shown in cyan) active site of the short ATP and self target RNA bound effector complex. The target RNA is shown in a stick representation with the bases shown in purple. The distance between the carboxylic acid hydroxyl of D32 (yellow) and the phosphate backbone is 4.2 Å. (D) A magnified view of the active site of Csm3.2 (shown in blue) of the non-self target RNA bound effector complex. The distance between the carboxylic acid hydroxyl of D32 (yellow) and the phosphate backbone is 10.2 Å. (E) A cartoon representation of Cas10 from the tall ATP bound effector complex with the HD domain shown in yellow, the first Palm domain shown in purple, the second Palm domain is shown in green cyan, and D4 is shown in split pea green. ATP1 and ATP2 are shown in a stick representation. (F) A magnified view of the highly conserved residues that are involved in interacting with ATP, including the GGDD motif shown as sticks in gray.

Figure 7.. Comparison of the S. epidermidis effector complexes with the equivalent complex from other organisms.

(A) Comparison of the atomic models of the short ATP and self target RNA bound S. epidermidis effector complex and the Type III-A effector complex from S. thermophilus (PDB ID: 6IFR). The atomic model of the S. epidermidis complex is shown on the left with the same color scheme as in Figure S6. The atomic model of the S. thermophilus complex is shown in the middle in with the color of each subunit shown in the figure. A superposition of the two structures is shown on the right. (B) Magnified views of the atomic model of the Csm3 active site of the Type III-A effector complex from S. epidermidis (left) and S. thermophilus (middle), and a superposition of the two (right). Both Csm3, crRNA, and target RNA models are shown in a cartoon representation with the bases shown as sticks in all panels. On the left the S. epidermidis Csm3 is shown in cyan. The crRNA in orange with the bases shown in green as sticks and the target RNA shown in orange with the bases shown in orange as sticks. The loop containing the active site residue D32 is labeled. in the middle the S. thermophilus Csm3 subunit is shown in red, the crRNA is shown in yellow, and the target RNA is shown in light pink. (C) A magnified view of the atomic model of Cas10 taken from the tall ATP bound effector complex. Palm 1 is shown in violet and Palm 2 is shown in green cyan. The two ATP molecules are shown in a stick representation. The conserved residues that are important for interacting with both ATP molecules are shown as sticks in gray. (D) A magnified view of the atomic model of non-self target RNA and ATP bound S. thermophilus Cas10 with the first Palm domain shown in purple and the second Palm domain shown in light blue. The ATP bound to the Cas10 observed in the cryo-EM structure is shown in a stick representation. The highly conserved residues that have been previously shown to be important for ATP binding are shown in as sticks in gray.