Extended Data Fig. 4: In-depth GsuCas4/Cas1-Cas2 interface analysis and structure-guided mutagenesis attempt to switch PAM specificity.
From: Mechanism for Cas4-assisted directional spacer acquisition in CRISPR–Cas
a. Overall dual-PAM structure. Insets: zoom-ins of interface between Cas4 and the two neighboring Cas1s. Cas4 connects to the non-catalytic Cas1 through a 20-amino acid fusion linker (colored in yellow), which mediates the dynamic docking and dissociation of Cas4. b. Surface electrostatic potential. Left inset: Cas2 contacts to the mid-duplex; Right inset: Cas1 end-stacking to the mid-duplex. Residues responsible for guiding the 3’-overhang are also shown. Cas1-Cas2 was found to specify a 22-bp mid-duplex rather than a 26-bp mid-duplex as defined by the integration assay; an additional two base-pairs are unwound from each end, and the mid-duplex is end-stacked by the N-terminal domain of the catalytic Cas1s on opposite ends. The 22-bp specification and the limited end-unwinding activity was previously observed in EfaCas1-Cas211,12. c. Cas1-Cas2 and Cas4-Cas2 interfaces. Top inset: the highly conserved C-terminus of Cas2 inserting into a hydrophobic pocket in Cas1, stabilizing complex formation. Bottom inset: the ceiling helix of Cas4 (aa 39–50) makes extensive polar contacts with a helix in Cas2 (aa 42–53). d. SEC, SDS-PAGE, and urea-PAGE analyses of the prespacer-bound complex used in cryo-EM analysis. They reveal the molecular weight, protein integrity, and prespacer integrity, respectively. For example, urea-PAGE reveals the PAM-overhang is not cleaved inside the Cas4/1-2 complex. e. Modeling the impact on PAM recognition by introducing the equivalent residues of E18 and S191 in P. fur Cas4 into G. sul Cas4 (E18Y and S191A substitutions). Specific atom changes in A-to-G switching (N6O substitution and N2 amine addition) are highlighted in colored balls. The steric clashes (lightening arrows) to PfuPAM (3’-GGN in the 3’-overhang) are expected to be partially relieved when substitutions are in place. f. Impact of E18Y and S191A substitutions on PAM cleavage activity. g. In vivo spacer acquisition assay results for the wild type and PAM-specificity Cas4 mutants. While E18Y/S191A Cas4 showed compromised Gsu-PAM (TTN) prespacer integration, it was able to support integration of Pfu-PAM (CCN) containing prespacers in vivo. N = 3 biological independent assays were analyzed by PCR and the band quantification revealed integration efficiency. Data presented as mean ± s.e.m.
