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.
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.
Rapid spread of ZIKA virus (ZIKV) and its association with severe birth defects have raised worldwide concern. Recent studies have shown that ZIKV retains its infectivity and remains structurally stable at temperatures up to 40 °C, unlike dengue and other flaviviruses. In spite of recent cryo-EM structures that showed similar architecture of ZIKA and dengue virus (DENV) E protein shells, little is known that makes ZIKV so temperature insensitive. Here, we attempt to unravel the molecular basis of greater thermal stability of ZIKV over DENV2 by executing atomistic molecular dynamics (MD) simulations on the viral E protein shells at 37 °C. Our results suggest that ZIKA E protein shell retains its structural integrity through stronger inter-raft communications facilitated by a series of electrostatic and H-bonding interactions among multiple inter-raft residues. In comparison, the DENV2 E protein shell surface was loosly packed that exhibited holes at all 3-fold vertices, in close agreement with another EM structure solved at 37 °C. The residue-level information obtained from our study could pave way for designing small molecule inhibitors and specific antibodies to inhibit ZIKV E protein assembly and membrane fusion.
Human
plasma cholesteryl ester transfer protein (CETP) mediates
the transfer of neutral lipids from antiatherogenic high-density lipoproteins
(HDLs) to proatherogenic low-density lipoproteins (LDLs). Recent cryo-electron
microscopy studies have suggested that CETP penetrates its N- and
C-terminal domains in HDL and LDL to form a ternary complex, which
facilitates the lipid transfer between different lipoproteins. Inhibition
of CETP lipid transfer activity has been shown to increase the plasma
HDL-C levels and, therefore, became an effective strategy for combating
cardiovascular diseases. Thus, understanding the molecular mechanism
of inhibition of lipid transfer through CETP is of paramount importance.
Recently reported inhibitors, torcetrapib and anacetrapib, exhibited
low potency in addition to severe side effects, which essentially
demanded a thorough knowledge of the inhibition mechanism. Here, we
employ steered molecular dynamics simulations to understand how inhibitors
interfere with the neutral lipid transfer mechanism of CETP. Our study
revealed that inhibitors physically occlude the tunnel posing a high
energy barrier for lipid transfer. In addition, inhibitors bring about
the conformational changes in CETP that hamper CE passage and expose
protein residues that disrupt the optimal hydrophobicity of the CE
transfer path. The atomic level details presented here could accelerate
the designing of safe and efficacious CETP inhibitors.
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