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Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes1. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate2,3. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop4,5. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands4,6,7. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination4,8,9,10,11,12,13,14,15,16. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5′-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process17,18.

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Figure 1: Foreign-DNA-bound Cascade structure.
Figure 2: PAM recognition by Cse1 subunit of Cascade.
Figure 3: Active guidance of the non-target DNA strand by Cascade.
Figure 4: Model for PAM-dependent directional R-loop formation in type I CRISPR-Cas system.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The structure factors and coordinates for Cascade–partial-R-loop structures with 10-nucleotide and 35-nucleotide non-target protospacer have been deposited in the Protein Data Bank (PDB) under accession numbers 5H9F and 5H9E, respectively.

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Acknowledgements

This work is supported by National Institutes of Health (NIH) grants GM102543 and GM086766 to A.K., GM097330 to S.B. and GM108888 to B.W. NE-CAT beamlines were supported by NIH grants P41 GM103403 and S10 RR029205. We thank G. Feigenson and J. Mallon for technical help, and I. Finkelstein, I. Price and A. Dolan for discussions.

Author information

Authors and Affiliations

Authors

Contributions

R.P.H. and A.K. designed the research, R.P.H. determined the structure and performed biochemical analyses, Y.X., F.D. and P.B.G.vE. contributed to biochemical analysis, K.R. assisted with diffraction data collection and processing, and B.W. and S.B. contributed to assay setup. R.P.H., B.W. and A.K. wrote the manuscript.

Corresponding author

Correspondence to Ailong Ke.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Electron density of nucleic acids in Cascade–dsDNA complex structure.

2FoFc electron density map (1.0σ). Nucleic acid strands are shown as sticks and coloured as previously indicated.

Extended Data Figure 2 Comparison between the Cascade–partial-R-loop crystal structure and the Cascade–full-R-loop EM reconstruction.

Rigid body docking of the partial-R-loop–Cascade crystal structure into the EM reconstruction (EMD-5929) of the full R-loop–Cascade illustrates a similar overall conformation, with a correlation value of 0.83.

Extended Data Figure 3 Conformational changes in Cascade upon partial R-loop formation.

Comparison of free (PDB 4TVX; in a darker shade) and partial R-loop-bound (this study, in lighter shade) Cascade. Arrows indicate the direction of the movement. The CTD of Cse1 pivots 30° about a hinge at the Cse1 NTD–CTD interface. The amplified motion at the tip of the Cse1-CTD slides with the Cse2 dimer ~12 Å relative to the Cas7 scaffold, and protrudes upwards into the R-loop. Cse1-NTD remains in the docked position, stabilized by the clamping of Cse1 L1 loop onto the exposed tri-nucleotide motif on crRNA 5′-handle.

Extended Data Figure 4 Local conformational rearrangement in Cse1 at the proposed Cas3-binding site.

a, Vector map showing global and local conformational changes in Cse1. Cse1-NTD (in cyan) undergoes moderate movement. Cse1-CTD (in green) swings about a pivoting point (Trp307) as part of the global conformational changes, The Cse1 NTD–CTD interface (in orange) undergoes considerable local conformational rearrangement upon partial R-loop formation. b, SDS–PAGE of wild-type and mutant Cascades used in mutagenesis. All mutants contained stoichiometric amounts of Cse1, except Trp307Ala, in which Cse1 failed to assemble. Uncropped gels are shown in Supplementary Fig. 1. c, Detailed structure rearrangement at Cse1 NTD–CTD interface, involving amino acids 300–326. Different colours are used to differentiate structural elements (amino acid numbers tabulated to the right). Partial R-loop-bound and free Cascade structures are rendered in solid and semi-transparent cartoons, respectively. d, Trp307 from Cse1-NTD rotates inside a hydrophobic socket at Cse1-CTD during the conformational change (free-Cascade-CTD rendered in a darker shade of green). Disruption of this interaction (Trp307Ala) caused Cse1 dissociation from Cascade. e, Docking of our structure into the cryoEM reconstruction of the crosslinked Cascade–dsDNA–Cas3 complex (EMD-5930). Cas3 density and the location of the local rearrangement in Cse1 are circled to highlight their proximity.

Extended Data Figure 5 Guided entry of dsDNA into Cascade.

A series of positively charged residues from Cas7.5, Cas7.6, Cas5e and Cse1 (dark blue surfaces) guide the dsDNA into Cascade and towards PAM recognition elements in Cse1-NTD.

Extended Data Figure 6 Influence of PAM−1 base-pair composition on Cascade binding affinity and Cas3 cleavage efficiency.

a, Inosine substitution of GNT−1 led to a mild twofold binding defect, suggesting that the N2 amine of GNT−1 is a minor determinant of specificity. b, Binding Kd values of E. coli Cascade for all four base-pair combinations at PAM−1. Kd values for 5′-ATG, ATA, ATC and ATT PAMs were determined to be ~10, 20, 40 and 100 nM, respectively. c, Cas3 cleavage efficiency was governed by the Cascade affinity for the corresponding PAM-containing target (5′-ATG > ATA >> ATT ≈ ATC). Cascade concentration above the Kd led to efficient Cas3 cleavage. Experiments were done in triplicate and representative results are shown. Uncropped gels are shown in Supplementary Fig. 1.

Extended Data Figure 7 Conservation of PAM recognition elements.

a, Sequence alignment of Cse1 proteins with known structures. Identical residues are highlighted in red, conserved residues in red text. DNA-contacting residues in the Cse1-NTD and -CTD are marked by cyan and green asterisks, respectively. The glycine loop and glutamine wedge are in blue and orange boxes, respectively. Residues mediating the Trp307 ball-and-socket interaction are marked with black asterisks. b, Structural alignment of Cse1 PAM recognition elements. Apo Cse1 from T. Thermus thermophilus (PDB 4AN8), Acidithiobacillus ferrooxidans (PDB 4H3T) and T. fusca (PDB 3WVO) were superimposed with the Cse1 in our partial R-loop-forming E. coli Cascade structure. Despite sequence variation, a glycine-rich loop is present in each Cse1 structure, and probably has a similar function to recognize PAM from the minor groove (left inset). The glutamine-wedge protrusion is highly conserved in 3D. Each wedge features a long side chain at the tip (right inset), which probably stacks underneath PAM in a similar fashion.

Extended Data Figure 8 Rationalization of PAM−2 and PAM−3 specificity using nucleotide substitution and modelling.

a, Modelling of alternative base pairs at PAM−2 suggests that only the N2 amine of GT−2 would cause steric clashes with Cα of Gly160 (bottom right quadrant), this amine therefore may serve as the anti-determinant for the rejection of GT–CNT at PAM−2. b, EMSAs demonstrating that removal of this amine in inosine substitution rescued the Cascade binding defect. c, Whereas Lys268Ala contained reduced affinity for the correct PAM, it still possessed strong discrimination against GT–CNT at PAM−3 (5′-CTG), suggesting the further presence of a mechanism to reject GT−3. d, Inosine substitution of GT−3 restored the Cascade binding Kd to ~40 nM, leading to the conclusion that the N2 amine of GT−3 is a minor determinant of specificity. Experiments were done in triplicate and representative results are shown. Uncropped gels are shown in Supplementary Fig. 1.

Extended Data Table 1 Oligonucleotides used in this study
Extended Data Table 2 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1, uncropped gel data. Part a is related to Fig. 2e. Part b is related to Fig. 2f. Part c is related to Fig. 3b. d, Related to Extended Data Fig. 4b. e, Related to Extended Data Fig. 6a. f, Related to Extended Data Fig. 6b. g, Related to Extended Data Fig. 6c. h, Related to Extended Data Fig. 8b. i, Related to Extended Data Fig. 8c. j, Related to Extended Data Fig. 8d. (PDF 508 kb)

Global structure of Cascade-dsDNA structure docked into EM reconstruction of full R-loop Cascade complex (EMD-5929).

Global structure of Cascade-dsDNA structure docked into EM reconstruction of full R-loop Cascade complex (EMD-5929). (MP4 18526 kb)

Conformational change between Cascade-apo (PDB: 4TVX) and Cascade-dsDNA structures.

The first half of the video compares the global conformational changes between the two structures. The second half focuses on the local conformational changes near the Cas3 binding site. The Cse1-CTD pivots around W307 in Cse1-NTD. The local structural elements around W307 in Cse1-NTD undergo rearrangement. The surface exposed β-hairpin (aa 316-326) melts away, the nearby α-helix rotates ˜90° at the Cse1-NTD/CTD interface. The altered surface landscape, together with the altered alignment between Cse1-NTD and Cse1-CTD, likely licenses Cas3 to bind. (MP4 29890 kb)

Conformational change between Cascade-ssDNA (PDB: 4QYZ) and Cascade-dsDNA structures.

Conformational change between Cascade-ssDNA (PDB: 4QYZ) and Cascade-dsDNA structures. (MP4 9127 kb)

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Hayes, R., Xiao, Y., Ding, F. et al. Structural basis for promiscuous PAM recognition in type I–E Cascade from E. coli. Nature 530, 499–503 (2016). https://doi.org/10.1038/nature16995

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