Binding of viral nuclear localization signal peptides to importin-α nuclear transport protein.

Using all-atom replica-exchange molecular dynamics simulations, we mapped the mechanisms of binding of the nuclear localization signal (NLS) sequence from Venezuelan equine encephalitis virus (VEEV) capsid protein to importin-α (impα) transport protein. Our objective was to identify the VEEV NLS sequence fragment that confers native, experimentally resolved binding to impα as well as to study associated binding energetics and conformational ensembles. The two selected VEEV NLS peptide fragments, KKPK and KKPKKE, show strikingly different binding mechanisms. The minNLS peptide KKPK binds non-natively and nonspecifically by adopting five diverse conformational clusters with low similarity to the x-ray structure 3VE6 of NLS-impα complex. Despite the prevalence of non-native interactions, the minNLS peptide still largely binds to the impα major NLS binding site. In contrast, the coreNLS peptide KKPKKE binds specifically and natively, adopting a largely homogeneous binding ensemble with a dominant, highly native-like conformational cluster. The coreNLS peptide retains most of native binding interactions, including π-cation contacts and a tryptophan cage. While KKPK binding is governed by a complex multistate free energy landscape featuring transitions between multiple binding poses, the coreNLS peptide free energy map is simple, exhibiting a single dominant native-like bound basin. We argue that the origin of the coreNLS peptide binding specificity is several electrostatic interactions formed by the two C-terminal amino acids, Lys10 and Glu11, with impα. The coreNLS sequence is then sufficient for native binding, but none of the amino acids flanking minNLS, including Lys10 and Glu11, are strictly necessary for the native pose. Our analyses indicate that the VEEV coreNLS sequence is virtually unique among human and viral proteins interacting with impα making it a potential target for VEEV-specific inhibitors.

Can Free Energy Perturbation Simulations Coupled with Replica-Exchange Molecular Dynamics Study Ligands with Distributed Binding Sites?

Free energy perturbation coupled with replica exchange with solute tempering (FEP/REST) offers a rigorous approach to compute relative free energy changes for ligands. To determine the applicability of FEP/REST for the ligands with distributed binding poses, we considered two alchemical transformations involving three putative inhibitors I0, I1, and I2 of the Venezuelan equine encephalitis virus nuclear localization signal sequence binding to the importin-α (impα) transporter protein. I0 → I1 and I0 → I2 transformations, respectively, increase or decrease the polarity of the parent molecule. Our objective was three-fold─(i) to verify FEP/REST technical performance and convergence, (ii) to estimate changes in binding free energy ΔΔG, and (iii) to determine the utility of FEP/REST simulations for conformational binding analysis. Our results are as follows. First, our FEP/REST implementation properly follows FEP/REST formalism and produces converged ΔΔG estimates. Due to ligand inherent unbinding, the better FEP/REST strategy lies in performing multiple independent trajectories rather than extending their length. Second, I0 → I1 and I0 → I2 transformations result in overall minor changes in inhibitor binding free energy, slightly strengthening the affinity of I1 and weakening that of I2. Electrostatic interactions dominate binding interactions, determining the enthalpic changes. The two transformations cause opposite entropic changes, which ultimately govern binding affinities. Importantly, we confirm the validity of FEP/REST free energy estimates by comparing them with our previous REST simulations, directly probing binding of three ligands to impα. Third, we established that FEP/REST simulations can sample binding ensembles of ligands. Thus, FEP/REST can be applied (i) to study the energetics of the ligand binding without defined poses and showing minor differences in affinities |ΔΔG| ≲ 0.5 kcal/mol and (ii) to collect ligand binding conformational ensembles.

Binding of Venezuelan Equine Encephalitis Virus Inhibitors to Importin-α Receptors Explored with All-Atom Replica Exchange Molecular Dynamics.

Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-ß, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.

Lysine Acetylation Changes the Mechanism of Aß25-35 Peptide Binding and Dimerization in the DMPC Bilayer.

The impact of Lys28 acetylation on Alzheimer's Aß peptide binding to the lipid bilayer has not been previously studied, either experimentally or computationally. To probe this common post-translational modification, we performed all-atom replica exchange molecular dynamics simulations targeting binding and aggregation of acetylated acAß25-35 peptide within the DMPC bilayer. Using the unmodified Aß25-35 studied previously as a reference, our results can be summarized as follows. First, Lys28 acetylation strengthens the Aß25-35 hydrophobic moment and consequently promotes the helical structure across the peptide extending it into the N-terminus. Second, because Lys28 acetylation disrupts electrostatic contact between Lys28 and lipid phosphate groups, it reduces the binding affinity of acAß25-35 peptides to the DMPC bilayer. Accordingly, although acetylation preserves the bimodal binding featuring a preferred inserted state and a less probable surface bound state, it decreases the stability of the former. Third, acetylation promotes acAß25-35 aggregation and eliminates monomers as thermodynamically viable species. More importantly, acAß25-35 retains as the most thermodynamically stable the inserted dimer with unique head-to-tail helical aggregation interface. However, due to enhanced helix structure, this dimer state becomes less stable and is less likely to propagate into higher order aggregates. Thus, acetylation is predicted to facilitate the formation of low-molecular-weight oligomers. Other post-translational modifications, including phosphorylation and oxidation, reduce helical propensity and have divergent impact on aggregation. Consequently, acetylation, when considered in its totality, has distinct consequences on Aß25-35 binding and aggregation in the lipid bilayer.

In silico Gene Set and Pathway Enrichment Analyses Highlight Involvement of Ion Transport in Cholinergic Pathways in Autism: Rationale for Nutritional Intervention.

Food is the primary human source of choline, an essential precursor to the neurotransmitter acetylcholine, which has a central role in signaling pathways that govern sensorimotor functions. Most Americans do not consume their recommended amount of dietary choline, and populations with neurodevelopmental conditions like autism spectrum disorder (ASD) may be particularly vulnerable to consequences of choline deficiency. This study aimed to identify a relationship between ASD and cholinergic signaling through gene set enrichment analysis and interrogation of existing database evidence to produce a systems biology model. In gene set enrichment analysis, two gene ontologies were identified as overlapping for autism-related and for cholinergic pathways-related functions, both involving ion transport regulation. Subsequent modeling of ion transport intensive cholinergic signaling pathways highlighted the importance of two genes with autism-associated variants: GABBR1, which codes for the gamma aminobutyric acid receptor (GABAB 1), and KCNN2, which codes for calcium-activated, potassium ion transporting SK2 channels responsible for membrane repolarization after cholinergic binding/signal transmission events. Cholinergic signal transmission pathways related to these proteins were examined in the Pathway Studio environment. The ion transport ontological associations indicated feasibility of a dietary choline support as a low-risk therapeutic intervention capable of modulating cholinergic sensory signaling in autism. Further research at the intersection of dietary status and sensory function in autism is warranted.