At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces sitespecific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg 2+ , unveiling its cardinal structural role. This activated Mg 2+ -bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.

Aspartate proteases are potential targets for various diseases, and many of their inhibitors are FDA-approved drugs. However, these peptidomimetic and reversibly bound drugs become ineffective upon prolonged use. Attempts have been made to design and synthesize various nonpeptidic epoxide-based irreversible inhibitors to combat the drug-resistance enigma.Here, we study the mechanism of epoxide ring opening in two widely studied aspartate proteases, HIV-1 protease and pepsin. Our results from QM/MM molecular dynamics show that the epoxide ring opening in aspartate proteases follow a two-step mechanism with the formation of an oxyanion intermediate, stabilized by a set of water molecules in the protein active site. These water molecules by virtue of "low-barrier hydrogen bonds" with the epoxide ring reduce the intrinsic reaction barrier while remaining structurally unperturbed, thus playing a cocatalytic role. We validated our results by reproducing the experimentally observed protease/pepsin−epoxide covalent complexes as end products. The observed stability of our oxyanion intermediate in a four-water-coordinated state is also consistent with the reported stable state of the hydroxide ion in water as OH − (H 2 O) 4 . Our study could pave the way for the design of new class "HIV protease irreversible inhibitors" from the acquired knowledge of the structures of intermediate and transition states traced during the explored reaction mechanism.

No abstract

HIV-1 protease (HIVPR) is an important drug target for combating AIDS. This enzyme is an aspartyl protease that is functionally active in its dimeric form. Nuclear magnetic resonance reports have convincingly shown that a pseudosymmetry exists at the HIVPR active site, where only one of the two aspartates remains protonated over the pH range of 2.5–7.0. To date, all HIVPR-targeted drug design strategies focused on maximizing the size–shape complementarity and van der Waals interactions of the small molecule drugs with the deprotonated, symmetric active site envelope of crystallized HIVPR. However, these strategies were ineffective with the emergence of drug resistant protease variants, primarily due to the steric clashes at the active site. In this study, we traced a specificity in the substrate binding motif that emerges primarily from the asymmetrical electrostatic potential present in the protease active site due to the uneven protonation. Our detailed results from atomistic molecular dynamics simulations show that while such a specific mode of substrate binding involves significant electrostatic interactions, none of the existing drugs or inhibitors could utilize this electrostatic hot spot. As the electrostatic is long-range interaction, it can provide sufficient binding strength without the necessity of increasing the bulkiness of the inhibitors. We propose that introducing the electrostatic component along with optimal fitting at the binding pocket could pave the way for promising designs that might be more effective against both wild type and HIVPR resistant variants.

CRISPR-Cas12a is a powerful RNA-guided genome-editing system, also emerging as a robust diagnostic tool that cleaves double-stranded DNA using only the RuvC domain. This opens an overarching question on how the spatially distant DNA target strand (TS) traverses toward the RuvC catalytic core. Here, continuous tens of microsecond-long molecular dynamics and free- energy simulations reveal that an ⍺-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the TS by anchoring the crRNA:TS hybrid and elegantly guiding the TS toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:TS hybrid toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the TS within the RuvC core by bending inward. Understanding of this cardinal process in the functioning of Cas12a will enrich fundamental knowledge and facilitate further engineering strategies for genome-editing.