Asymmetric Roles of Two Histidine Residues in Streptococcus pyogenes Cas9 Catalytic Domains upon Chemical Rescue

Abstract

CRISPR-Cas9 technology has been at the forefront of the field of biology. The Streptococcus pyogenes (SpyCas9) protein forms a complex with guide RNA and can recognize and cleave double-stranded DNA through hybridization based on 20 base pairings. SpyCas9 has two nuclease domains, HNH and RuvC, each of which cuts each DNA strand, and both contain critical histidine residues. Although previously reported crystal structures provide useful geometric information, the extent to which these residues functionally contribute to catalysis is unknown. Here, we mutated histidine residues on HNH and RuvC domains to alanine or glycine and attempted to rescue the enzymatic activity by adding the imidazole molecule, using an in vitro DNA cleavage assay. H840A and H840G exhibited rescued enzymatic activity on the HNH domain following imidazole addition, suggesting that H840 acts as a general base. We also tested various chemicals and found that the pK a of imidazole derivatives, and not their molecular shape, correlated with the rescue effect. In contrast, both H983A and H983G on the RuvC domain did not exhibit a rescue effect following imidazole addition. Our chemical rescue approach will provide crucial insight into understanding Cas9 catalysis, complementing structural analyses.

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“…This data was different from the studies of Furuhata et al. (the basicity) and Krishnan et al (introduction of IMs to Michael acceptor). , In addition, although the long distance between the IM and QM was shown, our study indicated that introduction of IMs might promote the Michael addition of CEL. Interestingly, a similar phenomenon also appeared in another Michael acceptor, CDDO, and its derivatives, which indicated that the strategy might be applicable to more Michael addition receptors …”

“…16−18 Interestingly, the previous study reported that the catalytic activity of SpyCas9 histidine mutants can be restored by the addition of imidazoles and substituted imidazoles (IMs). 18 Moreover, Krishnan et al found that the covalent binding between acrylonitrile (a Michael acceptor) and thiol could be enhanced once an aromatic heterocycle (such as imidazole, triazole, pyridine, etc.) was introduced as an electronwithdrawing group to the Michael acceptor.…”

“…Imidazole as a nitrogen heterocycle is an important component of histidine in proteins. Histidine is generally located in the catalytic domains of enzymes and is responsible for the catalysis of multiple biochemical reactions, as imidazole acts as not only as an acid/base but also a nucleophile. − Interestingly, the previous study reported that the catalytic activity of SpyCas9 histidine mutants can be restored by the addition of imidazoles and substituted imidazoles (IMs) . Moreover, Krishnan et al found that the covalent binding between acrylonitrile (a Michael acceptor) and thiol could be enhanced once an aromatic heterocycle (such as imidazole, triazole, pyridine, etc.)…”

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“…The target DNA strand complementary to spacer RNA was cut by the HNH nuclease domain and the non-target DNA strand by the RuvC nuclease domain to produce a blunt-ended double-strand breakage at 3 bp upstream to PAM [63,64]. Either D 10 A [58] or H 983 A [65] mutation destroyed the RuvC nuclease activity. On the other hand, D 839 A [66], H 840 A [58], and N 863 A [67] mutations could eliminate the HNH nuclease activity.…”

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“…For instance, Cas9 recognizes the PAM sequence NGG found directly downstream of the target DNA on the non-target strand (ANDERS et al, 2014). The cleaving properties of the RNA-guided endonucleases lie on two catalytic domains: HNH and RuvC, each cutting a DNA strand (FURUHATA; KATO, 2021). The CRISPR-Cas system goes as far as creating the DSBs in a targeted and precise manner.…”