Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Lambda-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase.

The allosteric inhibition of insulin-like growth factor receptor 1 kinase (IGF1RK) is a potential strategy to overcome selectivity barriers for targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives that have been reported as allosteric inhibitors of IGF1RK. We further studied the dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation: forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen-bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the positioning of chemical substituents with different pharmacophore features at the R1 indole ring influences molecular interactions and binding conformations of indole-butyl-amine derivatives and, hence, dramatically affects their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

Structural Models for a Series of Allosteric Inhibitors of IGF1R Kinase.

The allosteric inhibition of Insulin-like Growth Factor Receptor 1 Kinase (IGF1RK) is a potential strategy to overcome selectivity barriers in targeting receptor tyrosine kinases. We constructed structural models of a series of 12 indole-butyl-amine derivatives which have been reported as allosteric inhibitors of IGF1RK. We further studied dynamics and interactions of each inhibitor in the allosteric pocket via all-atom explicit-solvent molecular dynamics (MD) simulations. We discovered that a bulky carbonyl substitution at the R1 indole ring is structurally unfavorable for inhibitor binding in the IGF1RK allosteric pocket. Moreover, we found that the most potent derivative (termed C11) acquires a distinct conformation, forming an allosteric pocket channel with better shape complementarity and interactions with the receptor. In addition to a hydrogen bonding interaction with V1063, the cyano derivative C11 forms a stable hydrogen bond with M1156, which is responsible for its unique binding conformation in the allosteric pocket. Our findings show that the position of chemical substituents at the R1 indole ring with different pharmacophore features influences molecular interactions and binding conformations of the indole-butyl-amine derivatives, hence dramatically affecting their potencies. Our results provide a structural framework for the design of allosteric inhibitors with improved affinities and specificities against IGF1RK.

Quantitative Assessment of Energetic Contributions of Residues in a SARS-CoV-2 Viral Enzyme/Nanobody Interface.

The highly conserved protease enzyme from SARS-CoV-2 (MPro) is crucial for viral replication and is an attractive target for the design of novel inhibitory compounds. MPro is known to be conformationally flexible and has been stabilized in an extended conformation in a complex with a novel nanobody (NB2B4), which inhibits the dimerization of the enzyme via binding to an allosteric site. However, the energetic contributions of the nanobody residues stabilizing the MPro/nanobody interface remain unresolved. We probed these residues using all-atom MD simulations in combination with alchemical free energy calculations by studying the physical residue-residue interactions and discovered the role of hydrophobic and electrostatic interactions in stabilizing the complex. Specifically, we found via mutational analysis that three interfacial nanobody residues (Y59, R106, and L109) contributed significantly, two residues (L107 and P110) contributed moderately, and two residues (H112 and T113) contributed minimally to the overall binding affinity of the nanobody. We also discovered that the nanobody affinity could be enhanced via a charge-reversal mutation (D62R) that alters the local interfacial electrostatic environment of this residue in the complex. These findings are potentially useful in designing novel synthetic nanobodies as allosteric inhibitors of MPro.

Adenine Methylation Enhances the Conformational Flexibility of an RNA Hairpin Tetraloop.

The N6-methyladenosine modification is one of the most abundant post-transcriptional modifications in ribonucleic acid (RNA) molecules. Using molecular dynamics simulations and alchemical free-energy calculations, we studied the structural and energetic implications of incorporating this modification in an adenine mononucleotide and an RNA hairpin structure. At the mononucleotide level, we found that the syn configuration is more favorable than the anti configuration by 2.05 ± 0.15 kcal/mol. The unfavorable effect of methylation was due to the steric overlap between the methyl group and a nitrogen atom in the purine ring. We then probed the effect of methylation in an RNA hairpin structure containing an AUCG tetraloop, which is recognized by a "reader" protein (YTHDC1) to promote transcriptional silencing of long noncoding RNAs. While methylation had no significant conformational effect on the hairpin stem, the methylated tetraloop showed enhanced conformational flexibility compared to the unmethylated tetraloop. The increased flexibility was associated with the outward flipping of two bases (A6 and U7) which formed stacking interactions with each other and with the C8 and G9 bases in the tetraloop, leading to a conformation similar to that in the RNA/reader protein complex. Therefore, methylation-induced conformational flexibility likely facilitates RNA recognition by the reader protein.

Molecular basis for differential recognition of an allosteric inhibitor by receptor tyrosine kinases.

Understanding kinase-inhibitor selectivity continues to be a major objective in kinase drug discovery. We probe the molecular basis of selectivity of an allosteric inhibitor (MSC1609119A-1) of the insulin-like growth factor-I receptor kinase (IGF1RK), which has been shown to be ineffective for the homologous insulin receptor kinase (IRK). Specifically, we investigated the structural and energetic basis of the allosteric binding of this inhibitor to each kinase by combining molecular modeling, molecular dynamics (MD) simulations, and thermodynamic calculations. We predict the inhibitor conformation in the binding pocket of IRK and highlight that the charged residues in the histidine-arginine-aspartic acid (HRD) and aspartic acid-phenylalanine-glycine (DFG) motifs and the nonpolar residues in the binding pocket govern inhibitor interactions in the allosteric pocket of each kinase. We suggest that the conformational changes in the IGF1RK residues M1054 and M1079, movement of the ⍺C-helix, and the conformational stabilization of the DFG motif favor the selectivity of the inhibitor toward IGF1RK. Our thermodynamic calculations reveal that the observed selectivity can be rationalized through differences observed in the electrostatic interaction energy of the inhibitor in each inhibitor/kinase complex and the hydrogen bonding interactions of the inhibitor with the residue V1063 in IGF1RK that are not attained with the corresponding residue V1060 in IRK. Overall, our study provides a rationale for the molecular basis of recognition of this allosteric inhibitor by IGF1RK and IRK, which is potentially useful in developing novel inhibitors with improved affinity and selectivity.

Interactions and Transport of a Bioconjugated Peptide Targeting the Mitomembrane.

The Szeto-Schiller (SS) peptides are a subclass of cell-penetrating peptides that can specifically target mitochondria and mediate conditions caused by mitochondrial dysfunction. In this work, we constructed an iron-chelating SS peptide and studied its interaction with a mitochondrial-mimicking membrane using atomistic molecular dynamics (MD) simulations. We report that the peptide/membrane interaction is thermodynamically favorable, and the localization of the peptide to the membrane is driven by electrostatic interactions between the cationic residues and the anionic phospholipid headgroups. The insertion of the peptide into the membrane is driven by hydrophobic interactions between the aromatic side chains in the peptide and the lipid acyl tails. We also probed the translocation of the peptide across the membrane by applying nonequilibrium steered MD simulations and resolved the translocation pathway, free energy profile, and metastable states. We explored four distinct orientations of the peptide along the translocation pathway and found that one orientation was energetically more favorable than the other orientations. We tested a significantly slower pulling velocity on the most thermodynamically favorable system and compared metastable states during peptide translocation. We found that the peptide can optimize hydrophobic interactions with the membrane by having aromatic side chains interacting with the lipid acyl tails instead of forming π-π interactions with each other. The mechanistic insights emerging from our work will potentially facilitate improved peptide design with enhanced activity.

Porous Self-Assemblies Mediated by Dumbbell Particles as Cross-Linking Agents.

Self-assembly of colloidal particles is emerging as a promising approach for producing novel materials. These colloidal particles can be synthesized with protrusions (lobes) on their surfaces that allow the formation of porous structures with a wide range of applications. Using Langevin dynamics simulations, we studied self-assembly in the binary mixtures of lobed colloidal particles with variations in their lobe sizes to investigate the feasibility of using dumbbell particles (with two lobes) as cross-linkers to increase the porosity in self-assembled morphologies. Each binary system was formed by mixing the dumbbell particles with one of the following types of particles: trigonal planar (three lobes), tetrahedral (four lobes), trigonal bipyramidal (five lobes), and octahedral (six lobes). We observed that the lobe size on each particle can be tuned to favor the formation of random aggregates and spherical aggregates when the lobes are larger and well-ordered crystalline structures when the lobes are smaller. We also observed that these polydisperse systems form self-assembled structures characterized by porosities higher than those of the structures formed by the monodisperse systems. These results indicate that the lobe size is an important design feature that can be optimized to achieve desired structures with distinct morphologies and porosities, and the dumbbell particles are effective cross-linking agents to enhance the porosity in self-assembled structures.

Self-Assembly in an Experimentally Realistic Model of Lobed Patchy Colloids.

Colloids with lobed architectures have been shown to self-assemble into promising porous structures with potential biomedical applications. The synthesis of these colloids via experiments can be tuned to vary the number and the position of the lobes. However, the polydispersity involving the numbers, sizes, and the dispositions of lobes, that is often observed in particle designs, can significantly affect their self-assembled structures. In this work, we go beyond the uniform lobe size conditions commonly considered in molecular simulations, and probe the effect of polydispersity due to non-uniform lobe sizes by studying self-assembly in three experimentally observable designs of lobed particles (dumbbell, two lobes; trigonal planar, three lobes; and tetrahedral, four lobes), using coarse-grained Langevin dynamics simulations in the NVT ensemble. With increasing polydispersity, we observed the formation of a crystalline structure from a disordered state for the dumbbell system, and a loss of order in the crystalline structures for the trigonal planar system. The tetrahedral system retained a crystalline structure with only a minor loss in compactness. We observed that the effect of polydispersity on the self-assembled morphology of a given system can be minimized by increasing the number of lobes. The polydispersity in the lobe size may also be useful in tuning self-assemblies toward desired structures.

Structural and computational studies of HIV-1 RNA.

Viruses remain a global threat to animals, plants, and humans. The type 1 human immunodeficiency virus (HIV-1) is a member of the retrovirus family and carries an RNA genome, which is reverse transcribed into viral DNA and further integrated into the host-cell DNA for viral replication and proliferation. The RNA structures from the HIV-1 genome provide valuable insights into the mechanisms underlying the viral replication cycle. Moreover, these structures serve as models for designing novel therapeutic approaches. Here, we review structural data on RNA from the HIV-1 genome as well as computational studies based on these structural data. The review is organized according to the type of structured RNA element which contributes to different steps in the viral replication cycle. This is followed by an overview of the HIV-1 transactivation response element (TAR) RNA as a model system for understanding dynamics and interactions in the viral RNA systems. The review concludes with a description of computational studies, highlighting the impact of biomolecular simulations in elucidating the mechanistic details of various steps in the HIV-1's replication cycle.

Mechanism of Ligand Discrimination by the NMT1 Riboswitch.

Riboswitches are conserved functional domains in mRNA that almost exclusively exist in bacteria. They regulate the biosynthesis and transport of amino acids and essential metabolites such as coenzymes, nucleobases, and their derivatives by specifically binding small molecules. Due to their ability to precisely discriminate between different cognate molecules as well as their common existence in bacteria, riboswitches have become potential antibacterial drug targets that could deliver urgently needed antibiotics with novel mechanisms of action. In this work, we report the recognition mechanisms of four oxidization products (XAN, AZA, UAC, and HPA) generated during purine degradation by an RNA motif termed the NMT1 riboswitch. Specifically, we investigated the physical interactions between the riboswitch and the oxidized metabolites by computing the changes in the free energy on mutating key nucleobases in the ligand binding pocket of the riboswitch. We discovered that the electrostatic interactions are central to ligand discrimination by this riboswitch. The relative binding free energies of the mutations further indicated that some of the mutations can also strengthen the binding affinities of the ligands (AZA, UAC, and HPA). These mechanistic details are also potentially relevant in the design of novel compounds targeting riboswitches.

Coupling of conformational dynamics and inhibitor binding in the phosphodiesterase-5 family.

Phosphodiesterase-5 (PDE5) is responsible for regulating the concentration of the second messenger molecule cGMP by hydrolyzing it into 5'-GMP. PDE5 is implicated in erectile dysfunction and cardiovascular diseases. The substrate binding site in the catalytic domain of PDE5 is surrounded by several dynamic structural motifs (including the α14 helix, M-loop, and H-loop) that are known to switch between inactive and active conformational states via currently unresolved structural intermediates. We evaluated the conformational dynamics of these structural motifs in the apo state and upon binding of an allosteric inhibitor (evodiamine) or avanafil, a competitive inhibitor. We employed enhanced sampling-based replica exchange solute scaling (REST2) method, principal component analysis (PCA), time-lagged independent component analysis (tICA), molecular dynamics (MD) simulations, and well-tempered metadynamics simulations to probe the conformational changes in these structural motifs. Our results support a regulatory mechanism for PDE5, where the α14 helix alternates between an inward (lower activity) conformation and an outward (higher activity) conformation that is accompanied by the folding/unfolding of the α8' and α8″ helices of the H-loop. When the allosteric inhibitor evodiamine is bound to PDE5, the inward (inactive) state of the α14 helix is preferred, thus preventing substrate access to the catalytic site. In contrast, competitive inhibitors of PDE5 block catalysis by occupying the active site accompanied by stabilization of the outward conformation of the α14 helix. Defining the conformational dynamics underlying regulation of PDE5 activation will be helpful in rational design of next-generation small molecules modulators of PDE5 activity.

Role of Dynamics and Mutations in Interactions of a Zinc Finger Antiviral Protein with CG-rich Viral RNA.

Zinc finger antiviral protein (ZAP) is a host antiviral factor that selectively inhibits the replication of a variety of viruses. ZAP recognizes the CG-enriched RNA sequences and activates the viral RNA degradation machinery. In this work, we investigated the dynamics of a ZAP/RNA complex and computed the energetics of mutations in ZAP that affect its binding to the viral RNA. The crystal structure of a mouse-ZAP/RNA complex showed that RNA interacts with the zinc finger 2 (ZF2) and ZF3 domains. However, we found that due to the dynamic behavior of the single-stranded RNA, the terminal nucleotides C1 and G2 of RNA change their positions from the ZF3 to the ZF1 domain. Moreover, the electrostatic interactions between the zinc ions and the viral RNA provide further stability to the ZAP/RNA complex. We also provide structural and thermodynamic evidence for seven residue pairs (C1-Arg74, C1-Arg179, G2-Arg74, U3-Lys76, C4-Lys76, G5-Arg95, and U6-Glu204) that show favorable ZAP/RNA interactions, although these interactions were not observed in the ZAP/RNA crystal structure. Consistent with the observations from the mouse-ZAP/RNA crystal structure, we found that four residue pairs (C4-Lys89, C4-Leu90, C4-Tyr108, and G5-Lys107) maintained stable interactions in MD simulations. Based on experimental mutagenesis studies and our residue-level interaction analysis, we chose seven residues (Arg74, Lys76, Lys89, Arg95, Lys107, Tyr108, and Arg179) for individual alanine mutations. In addition, we studied mutations in those residues that are only observed in the crystal structures as interacting with RNA (Tyr98, Glu148, and Arg170). Out of these 10 mutations, we found that the Ala mutation in each of the five residues Arg74, Lys76, Lys89, Lys107, and Glu148 significantly reduced the binding affinity of ZAP to RNA.

Structural models of viral insulin-like peptides and their analogs.

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (µIR, µIGF1R, and µhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/µIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.

Epitaxial Self-Assembly of Interfaces of 2D Metal-Organic Frameworks for Electroanalytical Detection of Neurotransmitters.

This paper identifies the electrochemical properties of individual facets of anisotropic layered conductive metal-organic frameworks (MOFs) based on M3(2,3,6,7,10,11-hexahydroxytriphenylene)2 (M3(HHTP)2) (M = Co, Ni). The electroanalytical advantages of each facet are then applied toward the electrochemical detection of neurochemicals. By employing epitaxially controlled deposition of M3(HHTP)2 MOFs on electrodes, the contribution of the basal plane ({001} facets) and edge sites ({100} facets) of these MOFs can be individually determined using electrochemical characterization techniques. Despite having a lower observed heterogeneous electron transfer rate constant, the {001} facets of the M3(HHTP)2 systems prove more selective and sensitive for the detection of dopamine than the {100} facets of the same MOF, with the limit of detection (LOD) of 9.9 ± 2 nM in phosphate-buffered saline and 214 ± 48 nM in a simulated cerebrospinal fluid. Langmuir isotherm studies accompanied by all-atom MD simulations suggested that the observed improvement in performance and selectivity is related to the adsorption characteristics of analytes on the basal plane versus edge sites of the MOF interfaces. This work establishes that the distinct crystallographic facets of 2D MOFs can be used to control the fundamental interactions between analyte and electrode, leading to tunable electrochemical properties by controlling their preferential orientation through self-assembly.

Progress in Simulation Studies of Insulin Structure and Function.

Insulin is a peptide hormone known for chiefly regulating glucose level in blood among several other metabolic processes. Insulin remains the most effective drug for treating diabetes mellitus. Insulin is synthesized in the pancreatic ß-cells where it exists in a compact hexameric architecture although its biologically active form is monomeric. Insulin exhibits a sequence of conformational variations during the transition from the hexamer state to its biologically-active monomer state. The structural transitions and the mechanism of action of insulin have been investigated using several experimental and computational methods. This review primarily highlights the contributions of molecular dynamics (MD) simulations in elucidating the atomic-level details of conformational dynamics in insulin, where the structure of the hormone has been probed as a monomer, dimer, and hexamer. The effect of solvent, pH, temperature, and pressure have been probed at the microscopic scale. Given the focus of this review on the structure of the hormone, simulation studies involving interactions between the hormone and its receptor are only briefly highlighted, and studies on other related peptides (e.g., insulin-like growth factors) are not discussed. However, the review highlights conformational dynamics underlying the activities of reported insulin analogs and mimetics. The future prospects for computational methods in developing promising synthetic insulin analogs are also briefly highlighted.

Role of Mutations in Differential Recognition of Viral RNA Molecules by Peptides.

The conserved noncoding RNA elements in viral genomes interact with proteins to regulate various events during viral replication. We report studies on the recognition mechanisms of two helical peptides, namely, a native (Rev) peptide and a lab-evolved (RSG1.2) peptide, by a highly conserved viral RNA element from the human immunodeficiency virus 1 genome. Specifically, we investigated the physical interactions between the viral RNA molecule and helical peptides by computing free energy changes on mutating key amino acid residues involved in recognition of an internal loop in the viral RNA molecule.

Molecular interactions and inhibition of the SARS-CoV-2 main protease by a thiadiazolidinone derivative.

We report molecular interactions and inhibition of the main protease (MPro ) of SARS-CoV-2, a key enzyme involved in the viral life cycle. By using a thiadiazolidinone (TDZD) derivative as a chemical probe, we explore the conformational dynamics of MPro via docking protocols and molecular dynamics simulations in all-atom detail. We reveal the local and global dynamics of MPro in the presence of this inhibitor and confirm the inhibition of the enzyme with an IC50 value of 1.39 ± 0.22 µM, which is comparable to other known inhibitors of this enzyme.

Structures and interactions of insulin-like peptides from cone snail venom.

The venomous insulin-like peptides released by certain cone snails stimulate hypoglycemic shock to immobilize fish and catch the prey. Compared to human insulin (hIns), the cone snail insulins (Con-Ins) are typically monomeric and shorter in sequence, yet they exhibit moderate hIns-like biological activity. We have modeled six variants of Con-Ins (G3, K1, K2, T1A, T1B, and T2) and carried out explicit-solvent molecular dynamics (MD) simulations of eight types of insulins, two with known structures (hIns and Con-Ins-G1) and six Con-Ins with modeled structures, to characterize key residues of each insulin that interact with the truncated human insulin receptor (µIR). We show that each insulin/µIR complex is stable during explicit-solvent MD simulations and hIns interactions indicate the highest affinity for the "site 1" of IR. The residue contact maps reveal that each insulin preferably interacts with the αCT peptide than the L1 domain of IR. Through analysis of the average nonbonded interaction energy contribution of every residue of each insulin for the µIR, we probe the residues establishing favorable interactions with the receptor. We compared the interaction energy of each residue of every Con-Ins to the µIR and observed that γ-carboxylated glutamate (Gla), His, Thr, Tyr, Tyr/His, and Asn in Con-Ins are favorable substitutions for GluA4, AsnA21, ValB12, LeuB15, GlyB20, and ArgB22 in hIns, respectively. The identified insulin analogs, although lacking the last eight residues of the B-chain of hIns, bind strongly to µIR. Our findings are potentially useful in designing potent fast-acting therapeutic insulin.

Water Dynamics in a Peptide-appended Pillar[5]arene Artificial Channel in Lipid and Biomimetic Membranes.

Peptide-appended Pillar[5]arene (PAP) is an artificial water channel that can be incorporated into lipid and polymeric membranes to achieve high permeability and enhanced selectivity for angstrom-scale separations [Shen et al. Nat. Commun. 9:2294 (2018)]. In comparison to commonly studied rigid carbon nanotubes, PAP channels are conformationally flexible, yet these channels allow a high water permeability [Y. Liu and H. Vashisth Phys. Chem. Chem. Phys. 21:22711 (2019)]. Using molecular dynamics (MD) simulations, we study water dynamics in PAP channels embedded in biological (lipid) and biomimetic (block-copolymer) membranes to probe the effect of the membrane environment on water transport characteristics of PAP channels. We have resolved the free energy surface and local minima for water diffusion within the channel in each type of membrane. We find that water follows single file transport with low free-energy barriers in regions surroundings the central ring of the PAP channel and the single file diffusivity of water correlates with the number of hydrogen bonding sites within the channel, as is known for other sub-nm pore-size synthetic and biological water channels [Horner et al. Sci. Adv. 1:e1400083 (2015)].