Molecular dynamics in multidimensional space explains how mutations affect the association path of neomycin to a riboswitch.

Riboswitches are messenger RNA (mRNA) fragments binding specific small molecules to regulate gene expression. A synthetic N1 riboswitch, inserted into yeast mRNA controls the translation of a reporter gene in response to neomycin. However, its regulatory activity is sensitive to single-point RNA mutations, even those distant from the neomycin binding site. While the association paths of neomycin to N1 and its variants remain unknown, recent fluorescence kinetic experiments indicate a two-step process driven by conformational selection. This raises the question of which step is affected by mutations. To address this, we performed all-atom two-dimensional replica-exchange molecular dynamics simulations for N1 and U14C, U14C[Formula: see text], U15A, and A17G mutants, ensuring extensive conformational sampling of both RNA and neomycin. The obtained neomycin association and binding paths, along with multidimensional free-energy profiles, revealed a two-step binding mechanism, consisting of conformational selection and induced fit. Neomycin binds to a preformed N1 conformation upon identifying a stable upper stem and U-turn motif in the riboswitch hairpin. However, the positioning of neomycin in the binding site occurs at different RNA-neomycin distances for each mutant, which may explain their different regulatory activities. The subsequent induced fit arises from the interactions of the neomycin's N3 amino group with RNA, causing the G9 backbone to rearrange. In the A17G mutant, the critical C6-A17/G17 stacking forms at a closer RNA-neomycin distance compared to N1. These findings together with estimated binding free energies coincide with experiments and elucidate why the A17G mutation decreases and U15A enhances N1 activity in response to neomycin.

Varying molecular interactions explain aspects of crowder-dependent enzyme function of a viral protease.

Biochemical processes in cells, including enzyme-catalyzed reactions, occur in crowded conditions with various background macromolecules occupying up to 40% of cytoplasm's volume. Viral enzymes in the host cell also encounter such crowded conditions as they often function at the endoplasmic reticulum membranes. We focus on an enzyme encoded by the hepatitis C virus, the NS3/4A protease, which is crucial for viral replication. We have previously found experimentally that synthetic crowders, polyethylene glycol (PEG) and branched polysucrose (Ficoll), differently affect the kinetic parameters of peptide hydrolysis catalyzed by NS3/4A. To gain understanding of the reasons for such behavior, we perform atomistic molecular dynamics simulations of NS3/4A in the presence of either PEG or Ficoll crowders and with and without the peptide substrates. We find that both crowder types make nanosecond long contacts with the protease and slow down its diffusion. However, they also affect the enzyme structural dynamics; crowders induce functionally relevant helical structures in the disordered parts of the protease cofactor, NS4A, with the PEG effect being more pronounced. Overall, PEG interactions with NS3/4A are slightly stronger but Ficoll forms more hydrogen bonds with NS3. The crowders also interact with substrates; we find that the substrate diffusion is reduced much more in the presence of PEG than Ficoll. However, contrary to NS3, the substrate interacts more strongly with Ficoll than with PEG crowders, with the substrate diffusion being similar to crowder diffusion. Importantly, crowders also affect the substrate-enzyme interactions. We observe that both PEG and Ficoll enhance the presence of substrates near the active site, especially near catalytic H57 but Ficoll crowders increase substrate binding more than PEG molecules.

Peptide nucleic acid conjugates and their antimicrobial applications-a mini-review.

Peptide nucleic acid (PNA) is a nucleic acid mimic with high specificity and binding affinity to natural DNA or RNA, as well as resistance to enzymatic degradation. PNA sequences can be designed to selectively silence gene expression, which makes PNA a promising tool for antimicrobial applications. However, the poor membrane permeability of PNA remains the main limiting factor for its applications in cells. To overcome this obstacle, PNA conjugates with different molecules have been developed. This mini-review focuses on covalently linked conjugates of PNA with cell-penetrating peptides, aminosugars, aminoglycoside antibiotics, and non-peptidic molecules that were tested, primarily as PNA carriers, in antibacterial and antiviral applications. The chemistries of the conjugation and the applied linkers are also discussed.

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

Kinetic analysis of IFIT1 and IFIT5 interactions with different native and engineered RNAs and its consequences for designing mRNA-based therapeutics.

In response to foreign RNA, cellular antiviral mechanisms stimulate high expression of interferon-induced proteins with tetratricopeptide repeats (IFITs). Two members of the IFIT protein family, IFIT1 and IFIT5, are capable of binding the very terminal 5' end of mRNA. In eukaryotes, these mRNA termini contain a cap structure (m7GpppN, cap 0) that is often subjected to further modifications. Here, we performed a thorough examination of IFIT1 and IFIT5 binding to a wide spectrum of differently capped as well as fully uncapped mRNAs. The kinetic analysis of IFIT1 and IFIT5 interactions with mRNA ligands indicates that the cap structure modifications considerably influence the stability of IFIT1/RNA complexes. The most stable complexes were formed between IFIT1 and GpppG/A- and m7GpppG/A-RNAs. Unexpectedly, we found that NAD+- and NADH-capped RNAs associate with IFIT5 with kinetic parameters comparable to pppG-RNA. Finally, we measured interactions of IFIT1 with mRNAs bearing modified synthetic cap analogs that start to become the important tools in biotechnological and medicinal research. We found that incorporation of modified cap analogs to the RNA protects the latter, to a certain degree, from the translational inhibition caused by IFIT1 protein.

Correction: Extracellular loops of BtuB facilitate transport of vitamin B12 through the outer membrane of E. coli.

[This corrects the article DOI: 10.1371/journal.pcbi.1008024.].

Crowding affects structural dynamics and contributes to membrane association of the NS3/4A complex.

Using molecular dynamics simulations, we describe how crowded environments affect the internal dynamics and diffusion of the hepatitis C virus proteases NS3/4A. This protease plays a key role in viral replication and is successfully used as a target for antiviral treatment. The NS3 enzyme requires a peptide cofactor, called NS4A, with its central part interacting with the NS3 ß-sheet, and flexible, protruding terminal tails that are unstructured in water solution. The simulations describe the enzyme and water molecules at atomistic resolution, whereas crowders are modeled via either all-atom or coarse-grained models to emphasize different aspects of crowding. Crowders reflect the polyethylene glycol (PEG) molecules used in the experiments to mimic the crowded surrounding. A bead-shell model of folded coarse-grained PEG molecules considers mainly the excluded volume effect, whereas all-atom PEG models afford more protein-like crowder interactions. Circular dichroism spectroscopy experiments of the NS4A N-terminal tail show that a helical structure is formed in the presence of PEG crowders. The simulations suggest that crowding may assist in the formation of an NS4A helical fragment, positioned exactly where a transmembrane helix would fold upon the NS4A contact with the membrane. In addition, partially interactive PEGs help the NS4A N-tail to detach from the protease surface, thus enabling the process of helix insertion and potentially helping the virus establish a replication machinery needed to produce new viruses. Results point to an active role of crowding in assisting structural changes in disordered protein fragments that are necessary for their biological function.

Vitamin B12-peptide nucleic acids use the BtuB receptor to pass through the Escherichia coli outer membrane.

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B12, which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B12 enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B12-specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B12-PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B12. From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a ß-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB ß-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B12 transport system can be hijacked to enable PNA delivery to E. coli cells.

Extracellular loops of BtuB facilitate transport of vitamin B12 through the outer membrane of E. coli.

Vitamin B12 (or cobalamin) is an enzymatic cofactor essential both for mammals and bacteria. However, cobalamin can be synthesized only by few microorganisms so most bacteria need to take it up from the environment through the TonB-dependent transport system. The first stage of cobalamin import to E. coli cells occurs through the outer-membrane receptor called BtuB. Vitamin B12 binds with high affinity to the extracellular side of the BtuB protein. BtuB forms a ß-barrel with inner luminal domain and extracellular loops. To mechanically allow for cobalamin passage, the luminal domain needs to partially unfold with the help of the inner-membrane TonB protein. However, the mechanism of cobalamin permeation is unknown. Using all-atom molecular dynamics, we simulated the transport of cobalamin through the BtuB receptor embedded in an asymmetric and heterogeneous E. coli outer-membrane. To enhance conformational sampling of the BtuB loops, we developed the Gaussian force-simulated annealing method (GF-SA) and coupled it with umbrella sampling. We found that cobalamin needs to rotate in order to permeate through BtuB. We showed that the mobility of BtuB extracellular loops is crucial for cobalamin binding and transport and resembles an induced-fit mechanism. Loop mobility depends not only on the position of cobalamin but also on the extension of luminal domain. We provided atomistic details of cobalamin transport through the BtuB receptor showing the essential role of the mobility of BtuB extracellular loops. A similar TonB-dependent transport system is used also by many other compounds, such as haem and siderophores, and importantly, can be hijacked by natural antibiotics. Our work could have implications for future delivery of antibiotics to bacteria using this transport system.

Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates.

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

Molecular mechanisms for dynamic regulation of N1 riboswitch by aminoglycosides.

A synthetic riboswitch N1, inserted into the 5'-untranslated mRNA region of yeast, regulates gene expression upon binding ribostamycin and neomycin. Interestingly, a similar aminoglycoside, paromomycin, differing from neomycin by only one substituent (amino versus hydroxyl), also binds to the N1 riboswitch, but without affecting gene expression, despite NMR evidence that the N1 riboswitch binds all aminoglycosides in a similar way. Here, to explore the details of structural dynamics of the aminoglycoside-N1 riboswitch complexes, we applied all-atom molecular dynamics (MD) and temperature replica-exchange MD simulations in explicit solvent. Indeed, we found that ribostamycin and neomycin affect riboswitch dynamics similarly but paromomycin allows for more flexibility because its complex lacks the contact between the distinctive 6' hydroxyl group and the G9 phosphate. Instead, a transient hydrogen bond of 6'-OH with A17 is formed, which partially diminishes interactions between the bulge and apical loop of the riboswitch, likely contributing to riboswitch inactivity. In many ways, the paromomycin complex mimics the conformations, interactions, and Na+ distribution of the free riboswitch. The MD-derived interaction network helps understand why riboswitch activity depends on aminoglycoside type, whereas for another aminoglycoside-binding site, aminoacyl-tRNA site in 16S rRNA, activity is not discriminatory.

Conformational Changes of Anoplin, W-MreB1-9, and (KFF)3K Peptides near the Membranes.

Many peptides interact with biological membranes, but elucidating these interactions is challenging because cellular membranes are complex and peptides are structurally flexible. To contribute to understanding how the membrane-active peptides behave near the membranes, we investigated peptide structural changes in different lipid surroundings. We focused on two antimicrobial peptides, anoplin and W-MreB1-9, and one cell-penetrating peptide, (KFF)3K. Firstly, by using circular dichroism spectroscopy, we determined the secondary structures of these peptides when interacting with micelles, liposomes, E. coli lipopolysaccharides, and live E. coli bacteria. The peptides were disordered in the buffer, but anoplin and W-MreB1-9 displayed lipid-induced helicity. Yet, structural changes of the peptide depended on the composition and concentration of the membranes. Secondly, we quantified the destructive activity of peptides against liposomes by monitoring the release of a fluorescent dye (calcein) from the liposomes treated with peptides. We observed that only for anoplin and W-MreB1-9 calcein leakage from liposomes depended on the peptide concentration. Thirdly, bacterial growth inhibition assays showed that peptide conformational changes, evoked by the lipid environments, do not directly correlate with the antimicrobial activity of the peptides. However, understanding the relation between peptide structural properties, mechanisms of membrane disruption, and their biological activities can guide the design of membrane-active peptides.

Antibacterial Peptide Nucleic Acids-Facts and Perspectives.

Antibiotic resistance is an escalating, worldwide problem. Due to excessive use of antibiotics, multidrug-resistant bacteria have become a serious threat and a major global healthcare problem of the 21st century. This fact creates an urgent need for new and effective antimicrobials. The common strategies for antibiotic discovery are based on either modifying existing antibiotics or screening compound libraries, but these strategies have not been successful in recent decades. An alternative approach could be to use gene-specific oligonucleotides, such as peptide nucleic acid (PNA) oligomers, that can specifically target any single pathogen. This approach broadens the range of potential targets to any gene with a known sequence in any bacterium, and could significantly reduce the time required to discover new antimicrobials or their redesign, if resistance arises. We review the potential of PNA as an antibacterial molecule. First, we describe the physicochemical properties of PNA and modifications of the PNA backbone and nucleobases. Second, we review the carriers used to transport PNA to bacterial cells. Furthermore, we discuss the PNA targets in antibacterial studies focusing on antisense PNA targeting bacterial mRNA and rRNA.

Towards Catalytic Antibiotics: Redesign of Aminoglycosides To Catalytically Disable Bacterial Ribosomes.

The emergence of multidrug-resistant pathogens that are resistant to the majority of currently available antibiotics is a significant clinical problem. The development of new antibacterial agents and novel approaches is therefore extremely important. We set out to explore the potential of catalytic antibiotics as a new paradigm in antibiotics research. Herein, we describe our pilot study on the design, synthesis, and biological testing of a series of new derivatives of the natural aminoglycoside antibiotic neomycin B for their potential action as catalytic antibiotics. The new derivatives showed significant antibacterial activity against wild-type bacteria and were especially potent against resistant and pathogenic strains including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Selected compounds displayed RNase activity even though the activity was not as high and specific as we would have expected. On the basis of the observed chemical and biochemical data, along with the comparative molecular dynamics simulations of the prokaryotic rRNA decoding site, we postulate that the rational design of catalytic antibiotics should involve not only their structure but also a comprehensive analysis of the rRNA A-site dynamics.

Enzyme kinetics in crowded solutions from isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is a universal technique that directly measures the heat absorbed or released in a process. ITC is typically used to determine thermodynamic parameters of association of molecules without the need to label them. However, ITC is still rarely applied to study chemical reactions catalyzed by enzymes. In addition, these few studies of enzyme kinetic measurements that have been performed were in diluted solutions. Yet, to estimate realistic kinetic parameters, we have to account for the fact that enzymatic reactions in cells occur in a crowded environment because cells contain 200-400 g/L of macromolecular crowders such as proteins, ribosomes and lipids. Thus we expanded the ITC application for solutions mimicking the cellular environment by adding various macromolecular crowders. We investigated how these crowders affect the kinetics of trypsin-catalyzed reactions and determined the Michaelis-Menten parameters for hydrolysis of two trypsin substrates: Nα-benzoyl-l-arginine ethyl ester (BAEE) and Nα-benzoyl-dl-arginine ß-naphthylamide (BANA). Since ITC enables investigations of complex and turbid solutions with label-free reagents, it seems a perfect technique for kinetic analyses in crowded solutions. ITC also offers the opportunity to control enzyme-crowder and substrate-crowder interactions.

Enzymatic activity of human immunodeficiency virus type 1 protease in crowded solutions.

Cells are crowded with various macromolecules and metabolites, which affect biochemical reactions in many ways, from the diffusion of substrates to catalytic activities of enzymes. We herein investigated the proteolytic activity of the human immunodeficiency virus type 1 protease (HIV-1 PR) under non-crowded and crowded conditions. The latter environment was mimicked with various (poly)ethylene glycol molecules as crowding agents. We found that these crowding agents affect the kinetic parameters of the HIV-1 PR catalyzed reaction by increasing the Michaelis-Menten constant and decreasing the maximum velocity. The influence of crowding was concentration dependent. We explain this effect by the dynamics of the HIV-1 PR flexible flaps that cover the peptide substrate binding site and are crucial for enzyme activity, and by a possibly slower substrate-enzyme association time in the crowded conditions.

Computational Methods Used to Explore Transport Events in Biological Systems.

Transport of various molecules facilitated with membrane proteins is necessary for maintaining homeostasis in living cells. In humans, dysfunction of these proteins leads to many diseases. Thus, understanding how the membrane proteins function may help using them as therapeutic targets. To successfully investigate the mechanistic aspects of transport, the choice of appropriate methods is crucial. We review the computational methods that have proven most effective in investigating transport events, specifically, deterministic time-dependent classical molecular dynamics and its enhanced sampling variants, as well as methods based on Brownian dynamics. We describe technical aspects of these methods and examples of their novel variants or combinations that have been recently and successfully applied in the transport studies. We also discuss the difficulties related to these methods and provide possible solutions to avoid them.

d-Peptides as inhibitors of PR3-membrane interactions.

Proteinase 3 (PR3) is a neutrophil serine protease present in cytoplasmic granules but also expressed at the neutrophil surface where it mediates proinflammatory effects. Studies of the underlying molecular mechanisms have been hampered by the lack of inhibitors of the PR3 membrane anchorage. Indeed while there exist inhibitors of the catalytic activity of PR3, its membrane interfacial binding site (IBS) is distinct from its catalytic site. The IBS has been characterized both by mutagenesis experiments and molecular modeling. Through docking and molecular dynamics simulations we have designed d-peptides targeting the PR3 IBS. We used surface plasmon resonance to evaluate their effect on the binding of PR3 to phospholipid bilayers. Next, we verified their ability of binding to PR3 via fluorescence spectroscopy and isothermal titration calorimetry. The designed peptides did not affect the catalytic activity of PR3. A few peptides bound to PR3 hydrophobic pockets and inhibited PR3 binding to lipids. While the (KFF)3K d-peptide inconveniently showed a significant affinity for the lipids, another d-peptide (SAKEAFFKLLAS) did not and it inhibited the PR3-membrane binding site with IC50 of about 40µM. Our work puts forward d-peptides as promising inhibitors of peripheral protein-membrane interactions, which remain high-hanging fruits in drug design.

Does a Conjugation Site Affect Transport of Vitamin†B12 -Peptide Nucleic Acid Conjugates into Bacterial Cells?

Gram-negative bacteria develop specific systems for the uptake of scarce nutrients, including vitamin B12 . These uptake pathways may be utilized for the delivery of biologically relevant molecules into cells. Indeed, it was recently reported that vitamin B12 transported an antisense peptide nucleic acid (PNA) into Escherichia coli and Salmonella Typhimurium cells. The present studies indicate that the conjugation site of PNA to vitamin B12 has an impact on PNA transport into bacterial cells. Toward this end, a specifically designed PNA oligomer has been tethered at various positions of vitamin B12 (central Co, R5' -OH, c and e amide chains, meso position, and at the hydroxy group of cobinamide) by using known or newly developed methodologies and tested for the uptake of the synthesized conjugates by E. coli. Compounds in which the PNA oligonucleotide was anchored at the R5' -OH position were transported more efficiently than that of other compounds tethered at the peripheral positions around the corrin ring. Of importance is the fact that, contrary to mammalian organisms, E. coli also takes up cobinamide, which is an incomplete corrinoid. This selectivity opens up ways to fight bacterial infections.