HNH Nuclease Domain

The HNH nuclease domain (residues 775–908) lies in between the RuvC II–III motifs and forms only a few contacts with the rest of the protein. HNH nuclease domain comprises a two-stranded antiparallel β-sheet (β12 and β13) flanked by four α-helices (α35–α38). The HNH nuclease has a ββα-metal fold that comprises the active site, and there are many structural similarities between this nuclease and others that contain the ββα-metal fold, such as phage T4 endonuclease VII (Endo VII) and Vibrio vulnificus nuclease.

The HNH nuclease domain have three catalytic residues: Asp839, His840, and Asn863. His840 is critical for the cleavage of the complementary DNA strand, and Asn863 may point towards the active site and participate in catalysis. The Cas9 HNH domain cleaves the complementary strand of the target DNA through a single-metal mechanism, as observed for other HNH superfamily nucleases.

The crystal structures of Cas9 homologs, such as Streptococcus pyogenes Cas9 (SpyCas9), Francisella novicida Cas9 (FnCas9) and Staphylococcus aureus Cas9 (SaCas9) and so on, exhibit the HNH nuclease domains in the inactive conformations and the inactive state is located away from the complementary strand. The Cas9 HNH nuclease domain can approach the target DNA through conformational changes in the segment connecting the HNH and RuvC domains and cleave the complementary strand of the target DNA at a position three nucleotides upstream of the PAM sequence.

Dynamics of HNH Nuclease Domain Catalytic State

Despite recent advances in understanding Cas9 protein structures and its functional mechanism, little is known about the catalytic state of the Cas9 HNH nuclease domain and the key role of non-target DNA strand (ntDNA) in HNH repositioning. Identifying how the divalent metal ions affect the HNH nuclease domain conformational transition also remains elusive. A deeper understanding of Cas9 enzyme activation and its cleavage mechanism can enable further optimization of Cas9-based genome-editing specifcity and effciency. The derived catalytic conformation of the HNH domain can be exploited for rational engineering of Cas9 variants with enhanced specifcity.

Conformational activation of Cas9 HNH nuclease domain

Fig 1. Conformational activation of Cas9 HNH nuclease domain. (a) Apo autoinhibited state. (b) Complete DNA duplex bound pre-catalytic state. (c) Catalytically actine state.

Key Role of Non-target DNA Strand in HNH Nuclease Domain Repositioning

Recognition and cleavage of dsDNA strictly requires the presence of a protospacer adjacent motif (PAM) in the non-target DNA strand (ntDNA) and depends on the base-pair complementarity of the target DNA strand (tDNA) to the RNA guide template. The HNH domain samples larger conformational space in the absence of ntDNA. Binding of ntDNA likely stabilizes the intrinsically dynamic HNH nuclease domain in a closed conformation rather than in dynamic equilibrium between open and closed conformations. Therefore, omission of non-target DNA upstream of the PAM in the complex reconstitution most likely results in structures captured in an inactive conformation. The HNH domain samples a conformational equilibrium from an inactive state to an activated conformation, favoring the active state upon on-target ds DNA binding.

HNH makes very few contacts with the remainder of the Cas9 protein in other structures, whereas it interacts with Hel-II upon binding to dsDNA. This newly formed HNH–Hel2 intradomain interaction, formed by a hydrophobic surface patch and largely independent of crystal packing effects, appears to play an important role in effectively locking the HNH domain in an activated conformation for subsequent DNA strand scission and may function in a similar manner as the R-loop locking mechanism postulated for the type I Cascade surveillance complex.

Mg2+ Is Essential for Activation of HNH Nuclease Domain Catalytic State

The Mg2+ at the HNH Nuclease Domain catalytic center formed a favorable octahedral coordination with six surrounding oxygen atoms from different species. In addition to the three water molecules, the residues Asp839 and Asp861 on the ββα motif and the scissile phosphate between the nucleotides +3 and +4 of tDNA each contributes a coordination ligand. The residues play important role in stabilizing the Mg2+ cation.

His840 contributes marginally to Mg2+ binding, while the His840 side chain participates in a hydrogen-bond with a potential nucleophilic water molecule that is oriented for an in-line attack on the scissile bond. And Tyr823 and Arg864 appeared to play an important structural role in stabilizing the catalytic Asp839 side chain through hydrogen-bonding interactions. Primary sequence analysis shows that the tyrosine is strictly conserved among different types of CRISPR-Cas9, while the basic amino acid arginine (or lysine) is highly conserved among the Type II-A Cas9 orthologs.

To investigate whether Mg2+ could facilitate conformational activation of the HNH domain or not, scientists removed the coordinated Mg2+ from the catalytic conformation and performed microsecond-level conventional MD simulations. We first monitored the changes in the distance pair of +4P to His840 and to Asp861 at the cleavage interface. Their geometric mean increased from 6.0 Å in the catalytic state simulations to 10.5 Å on average, indicating detachment of the HNH domain from the tDNA. And the absence of Mg2+ leads the HNH domain to an intermediate state between the catalytic and pre-catalytic state. these results evidence that Mg2+ is essential for the formation and stability of the Cas9 HNH domain active state. Besides Mg2+, other metal ions like Mn2+, Ca2+ and Co2+ are also able to activate the HNH conformation and stabilize its catalytic state.

HNH Nuclease Domain Related Information

HNH Nuclease Domain Related References

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. Fuguo Jiang and Jennifer A. CRISPR–Cas9 Structures and Mechanisms. Annual Review of Biophysics. 2017 May 25. 46:505–29.
3. Jinek et al. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation. Science. 2014. 343(6176): 1247997–1247997.
4. Zhicheng Zuo and Jin Liu. Structure and Dynamics of Cas9 HNH Domain Catalytic State. Scientific Reports. 2017 December 08. 7: 17271.