RuvC domain consists of a six-stranded mixed β-sheet (β1, β2, β5, β11, β14 and β17) flanked by α-helices (α33, α34 and α39–α45) and two additional two-stranded antiparallel β-sheets (β3/β4 and β15/β16). The Cas9 RuvC domain comprises three split RuvC motifs (RuvC I–III), and interfaces with the PI domain to form a positively-charged surface that interacts with the 3′ tail of the sgRNA. Motifs II (residues 718–769) and III (residues 909–1098) are interrupted by the HNH domain, and motifs I (residues 1–59) and II are interrupted by a large lobe composed entirely of α-helices. This α-helical lobe, also referred to as the recognition (REC) lobe.
The RuvC domain have four catalytic residues: Asp10 (Ala), Glu762, His983 and Asp986. Asp10 is critical for the cleavage of the non-complementary DNA strand, and that Cas9 requires Mg2+ ions for the cleavage activity. Mutation of any of the four catalytic amino acids results in loss-of-function of the RuvC nuclease domain, and the creation of a functional Cas9 nickase. The catalytically competent state of the RuvC domain primed for cleaving the ntDNA, and Mg2+ is able to induce the formation of the active state for cleaving the ntDNA. The RuvC domain cleaves the non-complementary strand of the target DNA through the two-metal mechanism, as previously observed for other retroviral integrase superfamily nucleases.
Structural comparison of Cas9 nuclease domains to homologous structures of DNA-bound nucleases reveals that the Cas9 RuvC nuclease domain shares structural similarity with the retroviral integrase superfamily members characterized by an RNase H fold, such as Escherichia coli RuvC and Thermus thermophilus RuvC. But there are key structural dissimilarities between the Cas9 RuvC domain and the RuvC nucleases, which explain their functional differences. Unlike the Cas9 RuvC domain, the RuvC nucleases form dimers and recognize Holliday junctions. In addition to the conserved RNase H fold, the Cas9 RuvC domain has other structural elements involved in interactions with the guide: target heteroduplex (an end-capping loop between α42 and α43) and the PI domain/stem loop 3 (β-hairpin formed by β3 and β4).
Fig 1. Structure of the Cas9 RuvC nuclease domain
Fig 2. Two-metal mechanism of Cas9 RuvC nuclease domain
As is well known, Cas9 RuvC nuclease domain cleaves the non-complementary strand of the target DNA through the two-metal mechanism which was originally proposed to orient substrate, facilitate acid-base catalysis, and allow catalytic specificity to exceed substrate-binding specificity owing to the stringent metal-ion (Mg2+ in particular) coordination. Mg2+ Ions induce conformational changes to bridge the distance gap between the active site of the RuvC domain and the ntDNA. To prove this, Zuo and Liu conducted a series of experiments (Fig 2). S0 represents the simulation in the absence of Mg2+; S1/S2, and S5/S6 denote the repeated simulations in which the Mg2+ pair were positioned around − 3P and around −4P, respectively, whereas S3/S4 are the two simulations with the Mg2+ ions placed in between −3P and −4P. Without Mg2+ ions (S0), the distances between the ntDNA and the RuvC domain active residues peak at ~10 Å for both measurements.
Compared to the control simulation (S0), addition of the Mg2+ ions obviously shifted the distance distributions of ntDNA and RuvC active center to smaller values in varying degrees regardless of the Mg2+ ions positions (S1 to S6), indicating the presence of metal ions could indeed induce the conformational change toward the active state and close the distance gap between the ntDNA and the RuvC domain. Among all simulations, the double Mg2+ binding at the −4P in S5 and S6 leads to the largest reduction when comparing the highest peaks with that of S0. The observations uncover formation of an active state for ntDNA cleavage stemming from the binding of two Mg2+ at –4P.
The two Mg2+ binding at −4P is energetically more favorable than binding at other positions. Only a single Mg2+ is non-specially bound at the Cas9 RuvC active site, forming an incomplete coordination due to lack of the involvement of phosphate group. The presence of Mg2+ ions at −4P remarkably drive the RuvC domain active center to the opposite phosphate back-bone, leading to a reactant-like coordination primed for cleavage. Apart from its catalytic role, the second role of Mg2+ ions is to lead the inactive conformation of the RuvC domain and ntDNA toward the active state for catalysis. In summary, the results indicate that binding of double catalytic Mg2+ ions at RuvC domain catalytic center facilitates the formation of an active state for ntDNA cleavage, and, importantly, that Cas9-catalyzed tDNA cleavage produces 1-bp staggered ends rather than the blunt ends.
The feature of two-metal ion dependent nucleic acid enzymes, such as Cas9 RuvC nuclease domain, is an absolutely conserved Asp, which coordinates both metal ions and is replaced by a backbone phosphate in ribozymes. Basic residues in the active site of two-metal-ion enzymes are structurally and evolutionarily conserved. While among the one-metal ion dependent enzymes, there is no conserved Asp. The ββα-Me superfamily is marked by a conserved general base histidine, and the HUH enzymes by two His required for metal ion coordination. Furthermore, the difficulty level in capturing catalytically essential metal ions in crystal structures is another major difference. The two-metal ions are elusive even in enzyme-substrate complexes, but one-metal ion dependent enzymes can bind the metal ion in the absence of substrate.
Generally speaking, enzymes using two-metal ion catalysis are more stringent in metal ion selection and substrate specificity than one-metal ion-dependent enzymes. Mg2+, Ca2+, Mn2+, Zn2+, and Ni2+ etc, can support one-metal ion catalysis to varying degrees in many enzymes, while Mg2+ is typically required for two-metal mechanism. The preference for Mg2+ may root in the chemical environment and stringency imposed by coordination of two metal ions within 3–4 Å. The stringent requirement for double Mg2+ coordination is likely the basis for catalytic specificity by the two-metal ion mechanism. In contrast, catalytic specificity of one-metal ion dependent homing and HUH nucleases appears to derive from substrate binding.
1. Nishimasu et al. Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA. Cell. 2014 February 27; 156(5): 935–949.
2. Jinek et al. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science. 2014. 343(6176): 1247997–1247997.
3. Fuguo Jiang and Jennifer A. CRISPR–Cas9 Structures and Mechanisms. Annual Review of Biophysics. 2017 May 25. 46:505–29.
4. Yang. An Equivalent Metal Ion In One- and Two-metal Ion Catalysis. Nat Struct Mol Biol. Author manuscript; 2008 November ; 15(11): 1228–1231.
5. Zhicheng Zuo and Jin Liu. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations. Scientific Reports. 2016 November 22. 6:37584.