Energetic vs. entropic stabilization between a Remdesivir analogue and cognate ATP upon binding and insertion into the active site of SARS-CoV-2 RNA dependent RNA polymerase.

SARS-CoV-2 RNA dependent RNA polymerase (RdRp) serves as a highly promising antiviral drug target such as for a Remdesivir nucleotide analogue (RDV-TP or RTP). In this work, we mainly used alchemical all-atom simulations to characterize relative binding free energetics between the nucleotide analogue RTP and natural cognate substrate ATP upon initial binding and pre-catalytic insertion into the active site of SARS-CoV-2 RdRp. Natural non-cognate substrate dATP and mismatched GTP were also examined for computation control. We first identified significant differences in dynamical responses between nucleotide initial binding and subsequent insertion configurations to the open and closed active sites of the RdRp, respectively, though the RdRp protein conformational changes between the active site's open and closed states are subtle. Our alchemical simulations indicated that upon initial binding (active site open), RTP and ATP show similar binding free energies to the active sites while in the insertion state (active site closed), ATP is more stabilized (∼-2.4 kcal mol-1) than RTP in free energetics. Additional analyses show, however, that RTP is more stabilized in binding energetics than ATP, in both the insertion and initial binding states, with RTP more stabilized due to the electrostatic energy in the insertion state and due to vdW energy in the initial binding state. Hence, it appears that natural cognate ATP still excels at association stability with the RdRp active site due to that ATP maintains sufficient flexibilities e.g., in base pairing with the template, which exemplifies an entropic contribution to the cognate substrate stabilization. These findings highlight the importance of substrate flexibilities in addition to energetic stabilization in antiviral nucleotide analogue design.

Stability of H3O at extreme conditions and implications for the magnetic fields of Uranus and Neptune.

The anomalous nondipolar and nonaxisymmetric magnetic fields of Uranus and Neptune have long challenged conventional views of planetary dynamos. A thin-shell dynamo conjecture captures the observed phenomena but leaves unexplained the fundamental material basis and underlying mechanism. Here we report extensive quantum-mechanical calculations of polymorphism in the hydrogen-oxygen system at the pressures and temperatures of the deep interiors of these ice giant planets (to >600 GPa and 7,000 K). The results reveal the surprising stability of solid and fluid trihydrogen oxide (H3O) at these extreme conditions. Fluid H3O is metallic and calculated to be stable near the cores of Uranus and Neptune. As a convecting fluid, the material could give rise to the magnetic field consistent with the thin-shell dynamo model proposed for these planets. H3O could also be a major component in both solid and superionic forms in other (e.g., nonconvecting) layers. The results thus provide a materials basis for understanding the enigmatic magnetic-field anomalies and other aspects of the interiors of Uranus and Neptune. These findings have direct implications for the internal structure, composition, and dynamos of related exoplanets.

Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2.

Cas1 and Cas2 are highly conserved proteins across clustered-regularly-interspaced-short-palindromic-repeat-Cas systems and play a significant role in protospacer acquisition. Based on crystal structure of twofold symmetric Cas1-Cas2 in complex with dual-forked protospacer DNA (psDNA), we conducted all-atom molecular dynamics simulations to study the psDNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence, or PAMc, of Cas1-Cas2. In the simulation, we noticed that two active sites of Cas1 and Cas1' bind asymmetrically to two identical PAMc on the psDNA captured from the crystal structure. For the modified psDNA containing only one PAMc, as that to be recognized by Cas1-Cas2 in general, our simulations show that the non-PAMc association site of Cas1-Cas2 remains destabilized until after the stably bound PAMc being cleaved at the corresponding association site. Thus, long-range correlation appears to exist upon the PAMc cleavage between the two active sites (∼10 nm apart) on Cas1-Cas2, which can be allosterically mediated by psDNA and Cas2 and Cas2' in bridging. To substantiate such findings, we conducted repeated runs and further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high frequency in acquisition, respectively. Notably, such intersite correlation becomes even more pronounced for the Cas1-Cas2 in complex with psH but remains low for the Cas1-Cas2 in complex with psL. Hence, our studies demonstrate that PAMc recognition and cleavage at one active site of Cas1-Cas2 may allosterically regulate non-PAMc association or even cleavage at the other site, and such regulation can be mediated by noncatalytic Cas2 and DNA protospacer to possibly support the ensued psDNA acquisition.

Structural mechanism for guanylate-binding proteins (GBPs) targeting by the Shigella E3 ligase IpaH9.8.

The guanylate-binding proteins (GBPs) belong to the dynamin superfamily of GTPases and function in cell-autonomous defense against intracellular pathogens. IpaH9.8, an E3 ligase from the pathogenic bacterium Shigella flexneri, ubiquitinates a subset of GBPs and leads to their proteasomal degradation. Here we report the structure of a C-terminally truncated GBP1 in complex with the IpaH9.8 Leucine-rich repeat (LRR) domain. IpaH9.8LRR engages the GTPase domain of GBP1, and differences in the Switch II and α3 helix regions render some GBPs such as GBP3 and GBP7 resistant to IpaH9.8. Comparisons with other IpaH structures uncover interaction hot spots in their LRR domains. The C-terminal region of GBP1 undergoes a large rotation compared to previously determined structures. We further show that the C-terminal farnesylation modification also plays a role in regulating GBP1 conformation. Our results suggest a general mechanism by which the IpaH proteins target their cellular substrates and shed light on the structural dynamics of the GBPs.

Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control.

An elongation cycle of a transcribing RNA polymerase (RNAP) usually consists of multiple kinetics steps, so there exist multiple kinetic checkpoints where non-cognate nucleotides can be selected against. We conducted comprehensive free energy calculations on various nucleotide insertions for viral T7 RNAP employing all-atom molecular dynamics simulations. By comparing insertion free energy profiles between the non-cognate nucleotide species (rGTP and dATP) and a cognate one (rATP), we obtained selection free energetics from the nucleotide pre-insertion to the insertion checkpoints, and further inferred the selection energetics down to the catalytic stage. We find that the insertion of base mismatch rGTP proceeds mainly through an off-path along which both pre-insertion screening and insertion inhibition play significant roles. In comparison, the selection against dATP is found to go through an off-path pre-insertion screening along with an on-path insertion inhibition. Interestingly, we notice that two magnesium ions switch roles of leave and stay during the dATP on-path insertion. Finally, we infer that substantial selection energetic is still required to catalytically inhibit the mismatched rGTP to achieve an elongation error rate ∼10-4 or lower; while no catalytic selection seems to be further needed against dATP to obtain an error rate ∼10-2.

Balancing Non-Equilibrium Driving with Nucleotide Selectivity at Kinetic Checkpoints in Polymerase Fidelity Control.

High fidelity gene transcription and replication require kinetic discrimination of nucleotide substrate species by RNA and DNA polymerases under chemical non-equilibrium conditions. It is known that sufficiently large free energy driving force is needed for each polymerization or elongation cycle to maintain far-from-equilibrium to achieve low error rates. Considering that each cycle consists of multiple kinetic steps with different transition rates, one expects that the kinetic modulations by polymerases are not evenly conducted at each step. We show that accelerations at different kinetic steps impact quite differently to the overall elongation characteristics. In particular, for forward transitions that discriminate cognate and non-cognate nucleotide species to serve as kinetic selection checkpoints, the transition cannot be accelerated too quickly nor retained too slowly to obtain low error rates, as balancing is needed between the nucleotide selectivity and the non-equilibrium driving. Such a balance is not the same as the speed-accuracy tradeoff in which high accuracy is always obtained at sacrifice of speed. For illustration purposes, we used three-state and five-state models of nucleotide addition in the polymerase elongation and show how the non-equilibrium steady state characteristics change upon variations on stepwise forward or backward kinetics. Notably, by using the multi-step elongation schemes and parameters from T7 RNA polymerase transcription elongation, we demonstrate that individual transitions serving as selection checkpoints need to proceed at moderate rates in order to sustain the necessary non-equilibrium drives as well as to allow nucleotide selections for an optimal error control. We also illustrate why rate-limiting conformational transitions of the enzyme likely play a significant role in the error reduction.

Dissecting the CRISPR Cas1-Cas2 Protospacer Binding and Selection Mechanism by Using Molecular Dynamics Simulations.

Cas1 and Cas2 are highly conserved proteins among the clustered regularly interspaced short palindromic repeat Cas (CRISPR-Cas) systems and play a crucial role in protospacer selection and integration. According to the double-forked CRISPR Cas1-Cas2 complex, we conducted extensive all-atom molecular dynamics simulations to investigate the protospacer DNA binding and recognition. Our findings revealed that single-point amino acid mutations in Cas1 or in Cas2 had little impact on the binding of the protospacer, both in the binding and precatalytic states. In contrast, multiple-point amino acid mutations, particularly G74A, P80L, and V89A mutations on Cas2 and Cas2' proteins (m-multiple1 system), significantly affected the protospacer binding and selection. Notably, mutations on Cas2 and Cas2' led to an increased number of hydrogen bonds (#HBs) between Cas2&Cas2' and the dsDNA in the m-multiple1 system compared with the wild-type system. And the strong electrostatic interactions between Cas1-Cas2 and the protospacer DNA (psDNA) in the m-multiple1 system again suggested the increase in the binding affinity of protospacer acquisition. Specifically, mutations in Cas2 and Cas2' can remotely make the protospacer adjacent motif complementary (PAMc) sequences better in recognition by the two active sites, while multiple mutations K211E, P202Q, P212L, R138L, V134A, A286T, P282H, and P294H on Cas1a/Cas1b&Cas1a'/Cas1b' (m-multiple2 system) decrease the #HBs and the electrostatic interactions and make the PAMc worse in recognition compared with the wild-type system.

Dissecting nucleotide selectivity in viral RNA polymerases.

Designing antiviral therapeutics is of great concern per current pandemics caused by novel coronavirus or SARS-CoV-2. The core polymerase enzyme in the viral replication/transcription machinery is generally conserved and serves well for drug target. In this work we briefly review structural biology and computational clues on representative single-subunit viral polymerases that are more or less connected with SARS-CoV-2 RNA dependent RNA polymerase (RdRp), in particular, to elucidate how nucleotide substrates and potential drug analogs are selected in the viral genome synthesis. To do that, we first survey two well studied RdRps from Polio virus and hepatitis C virus in regard to structural motifs and key residues that have been identified for the nucleotide selectivity. Then we focus on related structural and biochemical characteristics discovered for the SARS-CoV-2 RdRp. To further compare, we summarize what we have learned computationally from phage T7 RNA polymerase (RNAP) on its stepwise nucleotide selectivity, and extend discussion to a structurally similar human mitochondria RNAP, which deserves special attention as it cannot be adversely affected by antiviral treatments. We also include viral phi29 DNA polymerase for comparison, which has both helicase and proofreading activities on top of nucleotide selectivity for replication fidelity control. The helicase and proofreading functions are achieved by protein components in addition to RdRp in the coronavirus replication-transcription machine, with the proofreading strategy important for the fidelity control in synthesizing a comparatively large viral genome.

A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control.

RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and fidelity control in the RNAP transcription elongation. Here we summarize our computational work from several recent publications to demonstrate first how T7 RNAP translocates via Brownian alike motions along DNA right after the catalytic product release. Then we show how the backward translocation motions are prevented at post-translocation upon successful nucleotide incorporation, which is also subject to stepwise nucleotide selection and acts as a pawl for "selective ratcheting". The structural dynamics and energetics features revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet working scenario of a prototypical transcription machine with sophisticated nucleotide selectivity for fidelity control. The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPs and some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machinery in higher organisms.