Dynamical Nonequilibrium Molecular Dynamics Simulations Identify Allosteric Sites and Positions Associated with Drug Resistance in the SARS-CoV-2 Main Protease.

The SARS-CoV-2 main protease (Mpro) plays an essential role in the coronavirus lifecycle by catalyzing hydrolysis of the viral polyproteins at specific sites. Mpro is the target of drugs, such as nirmatrelvir, though resistant mutants have emerged that threaten drug efficacy. Despite its importance, questions remain on the mechanism of how Mpro binds its substrates. Here, we apply dynamical nonequilibrium molecular dynamics (D-NEMD) simulations to evaluate structural and dynamical responses of Mpro to the presence and absence of a substrate. The results highlight communication between the Mpro dimer subunits and identify networks, including some far from the active site, that link the active site with a known allosteric inhibition site, or which are associated with nirmatrelvir resistance. They imply that some mutations enable resistance by altering the allosteric behavior of Mpro. More generally, the results show the utility of the D-NEMD technique for identifying functionally relevant allosteric sites and networks including those relevant to resistance.

Identification and validation of novel microtubule suppressors with an imidazopyridine scaffold through structure-based virtual screening and docking.

Targeting the colchicine binding site of α/ß tubulin microtubules can lead to suppression of microtubule dynamics, cell cycle arrest and apoptosis. Therefore, the development of microtubule (MT) inhibitors is considered a promising route to anticancer agents. Our approach to identify novel scaffolds as MT inhibitors depends on a 3D-structure-based pharmacophore approach and docking using three programs MOE, Autodock and BUDE (Bristol University Docking Engine) to screen a library of virtual compounds. From this work we identified the compound 7-(3-hydroxy-4-methoxy-phenyl)-3-(3-trifluoromethyl-phenyl)-6,7-dihydro-3H-imidazo[4,5-b]pyridin-5-ol (6) as a novel inhibitor scaffold. This compound inhibited several types of cancer cell proliferation at low micromolar concentrations with low toxicity. Compound 6 caused cell cycle arrest in the G2/M phase and blocked tubulin polymerization at low micromolar concentration (IC50 = 6.1 ±0.1 µM), inducing apoptosis via activation of caspase 9, increasing the level of the pro-apoptotic protein Bax and decreasing the level of the anti-apoptotic protein Bcl2. In summary, our approach identified a lead compound with potential antimitotic and antiproliferative activity.

ATP hydrolysis and nucleotide exit enhance maltose translocation in the MalFGK2E importer.

ATP binding cassette (ABC) transporters employ ATP hydrolysis to harness substrate translocation across membranes. The Escherichia coli MalFGK2E maltose importer is an example of a type I ABC importer and a model system for this class of ABC transporters. The MalFGK2E importer is responsible for the intake of malto-oligossacharides in E.coli. Despite being extensively studied, little is known about the effect of ATP hydrolysis and nucleotide exit on substrate transport. In this work, we studied this phenomenon using extensive molecular dynamics simulations (MD) along with potential of mean force calculations of maltose transport across the pore, in the pre-hydrolysis, post-hydrolysis and nucleotide-free states. We concluded that ATP hydrolysis and nucleotide exit trigger conformational changes that result in the decrease of energetic barriers to maltose translocation towards the cytoplasm, with a concomitant increase of the energy barrier in the periplasmic side of the pore, contributing for the irreversibility of the process. We also identified key residues that aid in positioning and orientation of maltose, as well as a novel binding pocket for maltose in MalG. Additionally, ATP hydrolysis leads to conformations similar to the nucleotide-free state. This study shows the contribution of ATP hydrolysis and nucleotide exit in the transport cycle, shedding light on ABC type I importer mechanisms.

A potential interaction between the SARS-CoV-2 spike protein and nicotinic acetylcholine receptors.

Changeux et al. (Changeux et al. C. R. Biol. 343:33-39.) recently suggested that the SARS-CoV-2 spike protein may interact with nicotinic acetylcholine receptors (nAChRs) and that such interactions may be involved in pathology and infectivity. This hypothesis is based on the fact that the SARS-CoV-2 spike protein contains a sequence motif similar to known nAChR antagonists. Here, we use molecular simulations of validated atomically detailed structures of nAChRs and of the spike to investigate the possible binding of the Y674-R685 region of the spike to nAChRs. We examine the binding of the Y674-R685 loop to three nAChRs, namely the human α4ß2 and α7 subtypes and the muscle-like αßγδ receptor from Tetronarce californica. Our results predict that Y674-R685 has affinity for nAChRs. The region of the spike responsible for binding contains a PRRA motif, a four-residue insertion not found in other SARS-like coronaviruses. The conformational behavior of the bound Y674-R685 is highly dependent on the receptor subtype; it adopts extended conformations in the α4ß2 and α7 complexes but is more compact when bound to the muscle-like receptor. In the α4ß2 and αßγδ complexes, the interaction of Y674-R685 with the receptors forces the loop C region to adopt an open conformation, similar to other known nAChR antagonists. In contrast, in the α7 complex, Y674-R685 penetrates deeply into the binding pocket in which it forms interactions with the residues lining the aromatic box, namely with TrpB, TyrC1, and TyrC2. Estimates of binding energy suggest that Y674-R685 forms stable complexes with all three nAChR subtypes. Analyses of simulations of the glycosylated spike show that the Y674-R685 region is accessible for binding. We suggest a potential binding orientation of the spike protein with nAChRs, in which they are in a nonparallel arrangement to one another.

Identification of the Initial Steps in Signal Transduction in the α4ß2 Nicotinic Receptor: Insights from Equilibrium and Nonequilibrium Simulations.

Nicotinic acetylcholine receptors (nAChRs) modulate synaptic transmission in the nervous system. These receptors have emerged as therapeutic targets in drug discovery for treating several conditions, including Alzheimer's disease, pain, and nicotine addiction. In this in silico study, we use a combination of equilibrium and nonequilibrium molecular dynamics simulations to map dynamic and structural changes induced by nicotine in the human α4ß2 nAChR. They reveal a striking pattern of communication between the extracellular binding pockets and the transmembrane domains (TMDs) and show the sequence of conformational changes associated with the initial steps in this process. We propose a general mechanism for signal transduction for Cys-loop receptors: the mechanistic steps for communication proceed firstly through loop C in the principal subunit, and are subsequently transmitted, gradually and cumulatively, to loop F of the complementary subunit, and then to the TMDs through the M2-M3 linker.

Molecular structure of FoxE, the putative iron oxidase of Rhodobacter ferrooxidans SW2.

The ancient metabolism of photoferrotrophy is likely to have played a key role in the biogeochemical cycle of iron on Early Earth leading to the deposition of Banded Iron Formations prior to the emergence of oxygenic photosynthesis. Extant organisms still performing this metabolism provide a convenient window to peer into its molecular mechanisms. Here we report the molecular structure of FoxE, the putative terminal iron oxidase of Rhodobacter ferrooxidans SW2. This protein is organized as a trimer with two hemes and a disulfide bridge per monomer. The distance between hemes, their solvent exposure and the surface electrostatics ensure a controlled electron transfer rate. They also guarantee segregation between electron capture from ferrous iron and electron release to downstream acceptors, which do not favor the precipitation of ferric iron. Combined with the functional characterization of this protein, the structure reveals how iron oxidation can be performed in the periplasmic space of this Gram-negative bacterium at circumneutral pH, while minimizing the risk of mineral precipitation and cell encrustation.

Inter-domain communication mechanisms in an ABC importer: a molecular dynamics study of the MalFGK2E complex.

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase domains is "transmitted" to the transmembrane domains. In this work, we focus our attention on the consequences of hydrolysis and inorganic phosphate exit in the maltose uptake system (MalFGK(2)E) from Escherichia coli. The prime goal is to identify and map the structural changes occurring during an ATP-hydrolytic cycle. For that, we use extensive molecular dynamics simulations to study three potential intermediate states (with 10 replicates each): an ATP-bound, an ADP plus inorganic phosphate-bound and an ADP-bound state. Our results show that the residues presenting major rearrangements are located in the A-loop, in the helical sub-domain, and in the "EAA motif" (especially in the "coupling helices" region). Additionally, in one of the simulations with ADP we were able to observe the opening of the NBD dimer accompanied by the dissociation of ADP from the ABC signature motif, but not from its corresponding P-loop motif. This work, together with several other MD studies, suggests a common communication mechanism both for importers and exporters, in which ATP-hydrolysis induces conformational changes in the helical sub-domain region, in turn transferred to the transmembrane domains via the "coupling helices".

Structural consequences of ATP hydrolysis on the ABC transporter NBD dimer: molecular dynamics studies of HlyB.

ABC transporters are a large and important family of membrane proteins involved in substrate transport across the membrane. The transported substrates are quite diverse, ranging from monatomic ions to large biomolecules. Consequently, some ABC transporters are involved in biomedically relevant situations, from genetic diseases to multidrug resistance. The most conserved domains in ABC transporters are the nucleotide binding domains (NBDs), which form a dimer responsible for the binding and hydrolysis of ATP, concomitantly with substrate translocation. To elucidate how ATP hydrolysis structurally affects the NBD dimer, and consequently the transporter, we performed a molecular dynamics study on the NBD dimer of the HlyB ABC exporter. We have observed a change in the contact surface between the monomers after hydrolysis, even though we have not seen dimer opening in any of the five 100 ns simulations. We have also identified specific regions that respond to ATP hydrolysis, in particular the X-loop motif of ABC exporters, which has been shown to be in contact with the coupling helices of the transmembrane domains (TMDs). We propose that this motif is an important part of the NBD-TMD communication in ABC exporters. Through nonequilibrium analysis, we have also identified gradual conformational changes within a short time scale after ATP hydrolysis.