Structure of Csm2 elucidates the relationship between small subunits of CRISPR‐Cas effector complexes

Abstract

Type I and type III CRISPR-Cas effector complexes share similar architecture and have homologous key subunits. However, the relationship between the so-called small subunits of these complexes remains a contentious issue. Here, it is shown that the recently solved structure of Thermotoga maritima Csm2 represents a dimer with the extensive structure swapping between monomers. Unswapping the structure generates a compact globular monomer which shares similar structure and surface properties with Cmr5, the small subunit of a related Cmr complex. Detailed analysis of available structures of small subunits reveals that they all have a common fold suggesting their common origin.

DNA Targeting by a Minimal CRISPR RNA-Guided Cascade

“…In both the apo- and target-bound Cascade structures, the top of Cas8c, located in the “belly” of the complex, can be easily segmented into three identical (cross-correlation coefficient of 0.97) sets of five alpha-helices (Figure 5A, left; Movie S2). This distinctive helical arrangement is structurally conserved in the small subunits of type I and type III surveillance complexes (Venclovas, 2016), confirming the bioinformatic prediction that Cas8c is a large subunit fused to a small subunit domain (Makarova et al, 2011a). The type III targeting complexes have three copies of the small subunit (Csm2 or Cmr5), while the type I-E targeting complex has two copies of its slightly larger small subunit (Cse2).…”

Structural Organization of a Type III-A CRISPR Effector Subcomplex Determined by X-ray Crystallography and Cryo-EM

“…Additionally, the crystal packing reveals that Loop 1 in each molecule faces the solvent space (Figure 2 -Figure supplement 1D). The second difference involves the third helix in the bundle, α3, which rearranges in SeCsm2 to form two helices, α4 and α5, whereas it is a single helix in an unswapped monomer of TmCsm2 (Figure 2B) (Venclovas, 2016). The predicted secondary structure of SeCsm2 indicates that α4-α5 should form a single helix (Figure 2 -Figure supplement 1C), which would be consistent with the helix topology described for Type III-A structures.…”

“…The overall structure of SeCsm2 reveals an exclusively α-helical fold composed of six helices (Figure 2A) forming a compact five-helix bundle with the sixth helix, α4, connecting two of the helices in the bundle. The organization of the helices generally follows the established fold for the small subunits of Class 1 CRISPR effector complexes (Gallo et al, 2016;Venclovas, 2016).…”

“…Instead, it breaks and kinks in the vicinity of residue 103. Interestingly, the occurrence of two helices at this position is more prevalent in the Type III-B small subunit, Cmr5 (Venclovas, 2016). Comparison of SeCsm2 α4-α5 to TmCsm2 α3 and α4-α5 from the Type III-B Thermus thermophilus HB8 Cmr5 structure (TtCmr5; PDB: 2ZOP) (Sakamoto et al, 2010) shows that SeCsm2 adopts an intermediate structural conformation between the single, linear helix in TmCsm2 and the pair of near perpendicular helices in TtCmr5 (Figure 2C).…”

“…SeCsm2 shows only two small positively charged electrostatic surfaces that could indicate a nucleic acid binding region (Figure 2 -Figure supplement 1F). In contrast, TtCmr5 exhibits a large positively charged electrostatic surface that has been implicated in target nucleic acid binding (Venclovas, 2016). When SeCsm2 and TtCmr5 are aligned, the positive patches are situated in different regions of the molecules, which may suggest alternate structural organizations of effector complex minor filaments.…”