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Influenza represents one of the biggest health threats facing humanity. Seasonal epidemics can transition to global pandemics, with cross-species infection presenting a continuous challenge. Although vaccines and several anti-viral options are available, constant genetic drifts and shifts vitiate any of the aforementioned prevention and treatment options. Therefore, we describe an approach targeted at the virus's channel to derive new anti-viral options. Specifically, Influenza A's M2 protein is a well-characterized channel targeted for a long time by aminoadamantane blockers. However, widespread mutations in the protein render the drugs ineffective. Consequently, we started by screening a repurposed drug library against aminoadamantane-sensitive and resistant M2 channels using bacteria-based genetic assays. Subsequent in cellulo testing and structure-activity relationship studies yielded a combination of Theobromine and Arainosine, which exhibits stark anti-viral activity by inhibiting the virus's channel. The drug duo was potent against H1N1 pandemic swine flu, H5N1 pandemic avian flu, aminoadamantane-resistant and sensitive strains alike, exhibiting activity that surpassed Oseltamivir, the leading anti-flu drug on the market. When this drug duo was tested in an animal model, it once more outperformed Oseltamivir, considerably reducing disease symptoms and viral RNA progeny. In conclusion, the outcome of this study represents a new potential treatment option for influenza alongside an approach that is sufficiently general and readily applicable to other viral targets.

An H-bond involves the sharing of a hydrogen atom between an electronegative atom to which it is covalently bound (the donor) and another electronegative atom serving as an acceptor. Such bonds represent a critically important geometrical force in biological macromolecules and, as such, have been characterized extensively. H-bond formation invariably leads to a weakening within the acceptor moiety due to the pulling exerted by the donor hydrogen. This phenomenon can be compared to a spring connecting two masses; pulling one mass stretches the spring, similarly affecting the bond between the two masses. Herein, we describe the opposite phenomenon when investigating the energetics of the C-H···O=C bond. This bond underpins the most prevalent protein transmembrane dimerization motif (GxxxG) in which a glycine Cα-H on one helix forms a hydrogen bond with a carbonyl in a nearby helix. We use isotope-edited FT-IR spectroscopy and corroborating computational approaches to demonstrate a surprising strengthening of the acceptor C=O bond upon binding with the glycine Cα-H. We show that electronic factors associated with the Cα-H bond strengthen the C=O oscillator by increasing the s -character of the σ-bond, lowering the hyperconjugative disruption of the π-bond. In addition, a reduction of the acceptor C=O bond's polarity is observed upon the formation of the C-H···O=C bond. Our findings challenge the conventional understanding of H-bond dynamics and provide new insights into the structural stability of inter-helical protein interactions.

Objective: Non-alcoholic fatty liver disease (NAFLD) involves hepatic accumulation of intracellular lipid droplets via incompletely understood processes. Here, we report distinct and cooperative NAFLD roles of LysTTT-5'tRF transfer RNA fragments and microRNA miR-194-5p., Methods: Combined use of diet induced obese mice with human-derived oleic acid-exposed Hep G2 cells revealed new NAFLD roles of LysTTT-5'tRF and miR-194-5p., Results: Unlike lean animals, dietary-induced NAFLD mice showed concurrent hepatic decrease of both LysTTT-5'tRF and miR-194-5p levels, which were restored following miR-132 antisense oligonucleotide treatment which suppresses hepatic steatosis. Moreover, exposing human-derived Hep G2 cells to oleic acid for 7 days co-suppressed miR-194-5p and LysTTT-5'tRF levels while increasing lipid accumulation. Inversely, transfecting fattened cells with a synthetic LysTTT-5'tRF mimic elevated mRNA levels of the metabolic regulator β-Klotho while decreasing triglyceride amounts by 30% within 24 h. In contradistinction, antisense suppression of miR-194-5p induced accumulation of its novel target, the NAFLD-implicated lipid droplet-coating PLIN2 protein. Further, two out of 15 steatosis-alleviating screened drug-repurposing compounds, Danazol and Latanoprost, elevated miR-194-5p or LysTTT-5'tRF levels., Conclusion: Our findings highlight the different yet complementary roles of miR-194-5p and LysTTT-5'tRF and offer new insights into the complex roles of small non-coding RNAs and the multiple pathways involved in NAFLD pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Mpox or monkeypox virus (MPXV) belongs to the subclass of Poxviridae and has emerged recently as a global threat. With a limited number of anti-viral drugs available for this new virus species, it is challenging to thwart the illness it begets. Therefore, characterizing new drug targets in the virus may prove advantageous to curbing the disease. Since channels as a family are excellent drug targets, we have sought to identify viral ion channels for this virus, which are instrumental in formulating channel-blocking anti-viral drugs. Bioinformatics analyses yielded eight transmembranous proteins smaller or equal to 100 amino acids in length. Subsequently, three independent bacteria-based assays have pointed to five of the eight proteins that exhibit ion channel activity. Finally, we propose a tentative structure of four ion channels from their primary amino acid sequences, employing AlphaFold2 and molecular dynamic simulation methods. These results may represent the first steps in characterizing MPXV viroporins en route to developing blockers that inhibit their function.

Detailed knowledge of a protein's key residues may assist in understanding its function and designing inhibitors against it. Consequently, such knowledge of one of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)'s proteins is advantageous since the virus is the etiological agent behind one of the biggest health crises of recent times. To that end, we constructed an exhaustive library of bacteria differing from each other by the mutated version of the virus's ORF3a viroporin they harbor. Since the protein is harmful to bacterial growth due to its channel activity, genetic selection followed by deep sequencing could readily identify mutations that abolish the protein's function. Our results have yielded numerous mutations dispersed throughout the sequence that counteract ORF3a's ability to slow bacterial growth. Comparing these data with the conservation pattern of ORF3a within the coronavirinae provided interesting insights: Deleterious mutations obtained in our study corresponded to conserved residues in the protein. However, despite the comprehensive nature of our mutagenesis coverage (108 average mutations per site), we could not reveal all of the protein's conserved residues. Therefore, it is tempting to speculate that our study unearthed positions in the protein pertinent to channel activity, while other conserved residues may correspond to different functionalities of ORF3a. In conclusion, our study provides important information on a key component of SARS-CoV-2 and establishes a procedure to analyze other viroporins comprehensively., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Flavivirus infections, such as those caused by dengue and West Nile viruses, emerge as new challenges for the global healthcare sector. It has been found that these two viruses encode ion channels collectively termed viroporins. Therefore, drug molecules that block such ion-channel activity can serve as potential antiviral agents and may play a primary role in therapeutic purposes. We screened 2839 FDA-approved drugs and compounds in advanced experimental phases using three bacteria-based channel assays to identify such ion channel blockers. We primarily followed a negative genetic screen in which the channel is harmful to the bacteria due to excessive membrane permeabilization that can be relieved by a blocker. Subsequently, we cross-checked the outcome with a positive genetic screen and a pH-dependent assay. The following drugs exhibited potential blocker activities: plerixafor, streptomycin, tranexamic acid, CI-1040, glecaprevir, kasugamycin, and mesna were effective against dengue virus DP1. In contrast, idasanutlin, benzbromarone, 5-azacytidine, and plerixafor were effective against West Nile Virus MgM. These drugs can serve as future antiviral therapeutic agents following subsequent in vitro and in vivo efficacy studies.

SARS-CoV-2 is the etiological agent COVID-19, one of the most impactful health crises afflicting humanity in recent decades. While research advances have yielded several treatment and prevention options, the pandemic is slow to abate, necessitating an expansion of our treatment arsenal. As a member of the coronaviridae, SARS-CoV-2 contains several ion channels, of which E and 3a are the best characterized. Since ion channels as a family are excellent drug targets, we sought to inhibit both viroporins as a means to curb infectivity. In a previous targeted study, we identified several blockers to each channel from an extensive drug repurposing library. Herein, we examined the ability of said compounds on the whole virus in cellulo. Gratifyingly, many of the blockers exhibited antiviral activity in a stringent assay examining protection from viral-driven death. In particular, darapladib and flumatinib, both 3a blockers, displayed potent antiviral activity. Furthermore, appreciable synergism between flumatinib and several E blockers was identified in a concentration regime in which the compounds are present in human plasma following oral administration. Taken together, targeting ion channels represents a promising approach to both augment and complement our antiviral arsenal against COVID-19.

Genus Flavivirus contains several important human pathogens. Among these, the Zika virus is an emerging etiological agent that merits concern. One of its structural proteins, prM, plays an essential role in viral maturation and assembly, making it an attractive drug and vaccine development target. Herein, we have characterized ZikV-M as a potential viroporin candidate using three different bacteria-based assays. These assays were subsequently employed to screen a library of repurposed drugs from which ten compounds were identified as ZikV-M blockers. Mutational analyses of conserved amino acids in the transmembrane domain of other flaviviruses, including West Nile and Dengue virus, were performed to study their role in ion channel activity. In conclusion, our data show that ZikV-M is a potential ion channel that can be used as a drug target for high throughput screening and drug repurposing.

Vibrational spectroscopy is a powerful tool used to analyze biological and chemical samples. However, in proteins, the most predominant peaks that arise from the backbone amide groups overlap one another, hampering site-specific analyses. Isotope editing has provided a robust, noninvasive approach to overcome this hurdle. In particular, the 1- 13 C═ 16 O and 1- 13 C═ 18 O labels that shift the amide I vibrational mode have enabled 1D- and 2D-IR spectroscopy to characterize proteins with excellent site-specific resolution. Herein, we expand the vibrational spectroscopy toolkit appreciably by introducing the 1- 13 C[Formula: see text] 15 N probe at specific locations along the protein backbone. A new, isotopically edited amide II peak is observed clearly in the spectra despite the presence of unlabeled modes arising from the rest of the protein. The experimentally determined shift of -30 cm -1 is reproduced by DFT calculations providing further credence to the mode assignment. Since the amide II mode arises from different elements than the amide I mode, it affords molecular insights that are both distinct and complementary. Moreover, multiple labeling schemes may be used simultaneously, enhancing vibrational spectroscopy's ability to provide detailed molecular insights.

SARS-CoV-2, the etiological agent of the COVID-19 pandemic, is a member of the Coronaviridae family. It is an enveloped virus with ion channels in its membrane, the most characterized of which is the E protein. Therefore, in an attempt to identify blockers of the E channel, we screened a library of 2839 approved-for-human-use drugs. Our approach yielded eight compounds that exhibited appreciable activity in three bacteria-based channel assays. Considering the fact that the E channel is the most conserved of all SARS-CoV-2 proteins, any inhibitor of its activity may provide an option to curb the viral spread. In addition, inhibitors can also enhance our ability to understand the exact role played by the E protein during the infectivity cycle. Finally, detailed electrophysiological analyses, alongside in vitro and in vivo studies will be needed to establish the exact potential of each of the blockers identified in our study.

Reproductive toxicity endpoints are a significant safety concern in the assessment of the adverse effects of chemicals in drug discovery. Computational models that can accurately predict a chemical's toxic potential are increasingly pursued to replace traditional animal experiments. Thus, ensemble learning models were built to predict the reproductive toxicity of compounds. Our ensemble models were developed using support vector machine, random forest, and extreme gradient boosting methods and 9 molecular fingerprints calculated for a dataset containing 1823 chemicals. The best prediction performance was achieved by the Ensemble-Top12 model, with an accuracy (ACC) of 86.33 %, a sensitivity (SEN) of 82.02 %, a specificity (SPE) of 90.19 %, and an area under the receiver operating characteristic curve (AUC) of 0.937 in 5-fold cross-validation and ACC, SEN, SPE, and AUC values of 84.38 %, 86.90 %, 90.67 %, and 0.920, respectively, in external validation. We also defined the applicability domain (AD) of the ensemble model by calculating the Tanimoto distance of the training set. Compared with models in existing literature, our ensemble model achieves relatively high ACC, SPE and AUC values. We also identified several fingerprint features related to chemical reproductive toxicity. Considering the performance of model, we recommend using the Ensemble-Top12 model to predict reproductive toxicity in early drug development., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The etiological agent of the COVID-19 pandemic is SARS-CoV-2. As a member of the Coronaviridae, the enveloped pathogen has several membrane proteins, of which two, E and 3a, were suggested to function as ion channels. In an effort to increase our treatment options, alongside providing new research tools, we have sought to inhibit the 3a channel by targeted drug repurposing. To that end, using three bacteria-based assays, we screened a library of 2839 approved-for-human-use drugs and identified the following potential channel-blockers: Capreomycin, Pentamidine, Spectinomycin, Kasugamycin, Plerixafor, Flumatinib, Litronesib, Darapladib, Floxuridine and Fludarabine. The stage is now set for examining the activity of these compounds in detailed electrophysiological studies and their impact on the whole virus with appropriate biosafety measures.

An important task in the early stage of drug discovery is the identification of mutagenic compounds. Mutagenicity prediction models that can interpret relationships between toxicological endpoints and compound structures are especially favorable. In this research, we used an advanced graph convolutional neural network (GCNN) architecture to identify the molecular representation and develop predictive models based on these representations. The predictive model based on features extracted by GCNNs can not only predict the mutagenicity of compounds but also identify the structure alerts in compounds. In fivefold cross-validation and external validation, the highest area under the curve was 0.8782 and 0.8382, respectively; the highest accuracy (Q) was 80.98% and 76.63%, respectively; the highest sensitivity was 83.27% and 78.92%, respectively; and the highest specificity was 78.83% and 76.32%, respectively. Additionally, our model also identified some toxicophores, such as aromatic nitro, three-membered heterocycles, quinones, and nitrogen and sulfur mustard. These results indicate that GCNNs could learn the features of mutagens effectively. In summary, we developed a mutagenicity classification model with high predictive performance and interpretability based on a data-driven molecular representation trained through GCNNs.

The correct balance between attractive, repulsive and peptide hydrogen bonding interactions must be attained for proteins to fold correctly. To investigate these important contributors, we sought a comparison of the folding between two 25-residues peptides, the influenza A M2 protein transmembrane domain (M2TM) and the 25-Ala (Ala 25 ). M2TM forms a stable α-helix as is shown by circular dichroism (CD) experiments. Molecular dynamics (MD) simulations with adaptive tempering show that M2TM monomer is more dynamic in nature and quickly interconverts between an ensemble of various α-helical structures, and less frequently turns and coils, compared to one α-helix for Ala 25 . DFT calculations suggest that folding from the extended structure to the α-helical structure is favored for M2TM compared with Ala 25 . This is due to CH⋯O attractive interactions which favor folding to the M2TM α-helix, and cannot be described accurately with a force field. Using natural bond orbital (NBO) analysis and quantum theory atoms in molecules (QTAIM) calculations, 26 CH⋯O interactions and 22 NH⋯O hydrogen bonds are calculated for M2TM. The calculations show that CH⋯O hydrogen bonds, although individually weaker, have a cumulative effect that cannot be ignored and may contribute as much as half of the total hydrogen bonding energy, when compared to NH⋯O, to the stabilization of the α-helix in M2TM. Further, a strengthening of NH⋯O hydrogen bonding interactions is calculated for M2TM compared to Ala 25 . Additionally, these weak CH⋯O interactions can dissociate and associate easily leading to the ensemble of folded structures for M2TM observed in folding MD simulations., (© 2020 Wiley Periodicals LLC.)

COVID-19 is one of the most impactful pandemics in recorded history. As such, the identification of inhibitory drugs against its etiological agent, SARS-CoV-2, is of utmost importance, and in particular, repurposing may provide the fastest route to curb the disease. As the first step in this route, we sought to identify an attractive and viable target in the virus for pharmaceutical inhibition. Using three bacteria-based assays that were tested on known viroporins, we demonstrate that one of its essential components, the E protein, is a potential ion channel and, therefore, is an excellent drug target. Channel activity was demonstrated for E proteins in other coronaviruses, providing further emphasis on the importance of this functionally to the virus' pathogenicity. The results of a screening effort involving a repurposing drug library of ion channel blockers yielded two compounds that inhibit the E protein: Gliclazide and Memantine. In conclusion, as a route to curb viral virulence and abate COVID-19, we point to the E protein of SARS-CoV-2 as an attractive drug target and identify off-label compounds that inhibit it., Competing Interests: Declaration of competing interest The authors declare that they are in the process of filing a patent for second medicinal use of Gliclazide and Memantine as anti COVID-19 agents., (Copyright © 2020 Elsevier Inc. All rights reserved.)

H-bonding is the predominant geometrical determinant of biomolecular structure and interactions. As such, considerable analyses have been undertaken to study its detailed energetics. The focus, however, has been mostly reserved for H-bonds comprising a single donor and a single acceptor. Herein, we measure the prevalence and energetics of multiplex H-bonds that are formed between three or more groups. We show that 92% of all transmembrane helices have at least one non-canonical H-bond formed by a serine or threonine residue whose hydroxyl side chain H-bonds to an over-coordinated carbonyl oxygen at position i -4, i -3, or i in the sequence. Isotope-edited FTIR spectroscopy, coupled with DFT calculations, enables us to determine the bond enthalpies, pointing to values that are up to 127% higher than that of a single canonical H-bond. We propose that these strong H-bonds serve to stabilize serine and threonine residues in hydrophobic environments while concomitantly providing them flexibility between different configurations, which may be necessary for function.

Viroporins are a family of small hydrophobic proteins found in many enveloped viruses that are capable of ion transport. Building upon the ability to inhibit influenza by blocking its archetypical M2 H + channel, as a family, viroporins may represent a viable target to curb viral infectivity. To this end, using three bacterial assays we analyzed six small hydrophobic proteins from biomedically important viruses as potential viroporin candidates. Our results indicate that Eastern equine encephalitis virus 6k, West Nile virus MgM, Dengue virus 2k, Dengue virus P1, Variola virus gp170, and Variola virus gp151 proteins all exhibit channel activity in the bacterial assays, and as such may be considered viroporin candidates. It is clear that more studies, such as patch clamping, will be needed to characterize the ionic conductivities of these proteins. However, our approach presents a rapid procedure to analyze open reading frames in other viruses, yielding new viroporin candidates for future detailed investigation. Finally, if conductivity is proven vital to their cognate viruses, the bio-assays presented herein afford a simple approach to screen for new channel blockers.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

The M2 protein is an important target for drugs in the fight against the influenza virus. Because of the emergence of resistance against antivirals directed toward the M2 proton channel, the search for new drugs against resistant M2 variants is of high importance. Robust and sensitive assays for testing potential drug compounds on different M2 variants are valuable tools in this search for new inhibitors. In this work, we describe a fluorescence sensor-based assay, which we termed "pHlux", that measures proton conduction through M2 when synthesized from an expression vector in Escherichia coli. The assay was compared to a previously established bacterial potassium ion transport complementation assay, and the results were compared to simulations obtained from analysis of a computational model of M2 and its interaction with inhibitor molecules. The inhibition of M2 was measured for five different inhibitors, including Rimantadine, Amantadine, and spiro type compounds, and the drug resistance of the M2 mutant variants (swine flu, V27A, and S31N) was confirmed. We demonstrate that the pHlux assay is robust and highly sensitive and shows potential for high-throughput screening.

Hydrogen exchange is a powerful method to examine macromolecules. In membrane proteins, exchange can distinguish between solvent-accessible and -inaccessible residues due to shielding by the hydrophobic environment of the lipid bilayer. Herein, rather than examining which residues undergo hydrogen exchange, we employ a protocol that enables the full deuteration of all polar hydrogens in a membrane protein. We then measure the impact of hydrogen exchange on the shift of the amide I vibrational mode of individually labeled sites. The results enable us to correlate polarity with vibrational shifts, thereby providing a powerful tool to examine specific locations within a membrane protein in its native membrane environment.

The development of drug resistance has long plagued our efforts to curtail viral infections in general and influenza in particular. The problem is particularly challenging since the exact mode of resistance may be difficult to predict, without waiting for untreatable strains to evolve. Herein, a different approach is taken. Using a novel genetic screen, we map the resistance options of influenza's M2 channel against its aminoadamantane antiviral inhibitors. In the process, we could identify clinically known resistant mutations in a completely unbiased manner. Additionally, novel mutations were obtained, which, while known to exist in circulating viruses, were not previously classified as drug resistant. Finally, we demonstrated the approach against an anti-influenza drug that has not seen clinical use, identifying several resistance mutations in the process. In conclusion, we present and employ a method to predict the resistance options of influenza's M2 channel to antiviral agents ahead of clinical use and without medical hazard., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In mammals, the apical sodium-dependent bile acid transporter (ASBT) is responsible for the reuptake of bile acid from the intestine, thus recycling bile acid that is secreted from the gallbladder, for the purpose of digestion. As bile acid is synthesized from cholesterol, ASBT inhibition could have important implications in regulation of cholesterol levels in the blood. We report on a simulation study of the recently resolved structures of the inward-facing ASBT from Neisseria meningitidis and from Yersinia frederiksenii, as well as of an ASBT variant from Yersinia frederiksenii suggested to be in the outward-facing conformation. Classical and steered atomistic simulations and comprehensive potential of mean force analyses of ASBT, both in the absence and presence of ions and substrate, allow us to characterize and gain structural insights into the Na(+) binding sites and propose a mechanistic model for the transport cycle. In particular, we investigate structural features of the ion translocation pathway, and suggest a third putative Na(+) binding site. Our study sheds light on the structure-function relationship of bacterial ASBT and may promote a deeper understanding of transport mechanism altogether., (© 2015 Wiley Periodicals, Inc.)

HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to possess ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K(+) transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K(+) concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers.

Solving structures of membrane proteins has always been a formidable challenge, yet even upon success, the results are normally obtained in a mimetic environment that can be substantially different from a biological membrane. Herein, we use noninvasive isotope-edited FTIR spectroscopy to derive a structural model for the SARS coronavirus E protein transmembrane domain in lipid bilayers. Molecular-dynamics-based structural refinement, incorporating the IR-derived orientational restraints points to the formation of a helical hairpin structure. Disulfide cross-linking and X-ray reflectivity depth profiling provide independent support of the results. The unusually short helical hairpin structure of the protein might explain its ability to deform bilayers and is reminiscent of other peptides with membrane disrupting functionalities. Taken together, we show that isotope-edited FTIR is a powerful tool to analyze small membrane proteins in their native environment, enabling us to relate the unusual structure of the SARS E protein to its function.

The Influenza Matrix 2 (M2) protein is the target of Amantadine and Rimantadine which block its H(+) channel activity. However, the potential of these aminoadamantyls to serve as anti-flu agents is marred by the rapid resistance that the virus develops against them. Herein, using a cell based assay that we developed, we identify two new aminoadamantyl derivatives that show increased activity against otherwise resistant M2 variants. In order to understand the distinguishing binding patterns of the different blockers, we computed the potential of mean force of the drug binding process. The results reveal that the new derivatives are less mobile and bind to a larger pocket in the channel. Finally, such analyses may prove useful in designing new, more effective M2 blockers as a means of curbing influenza. This article is part of a Special Issue entitled: Viral Membrane Proteins - Channels for Cellular Networking., (© 2013.)

Macromolecules are characterized by their particular arrangement of H bonds. Many of these interactions involve a single donor and acceptor pair, such as the regular H-bonding pattern between carbonyl oxygens and amide H(+)s four residues apart in α-helices. The H-bonding potential of some acceptors, however, leads to the phenomenon of overcoordination between two donors and one acceptor. Herein, using isotope-edited Fourier transform infrared measurements and density functional theory (DFT) calculations, we measured the strength of such bifurcated H bonds in a transmembrane α-helix. Frequency shifts of the (13)C=(18)O amide I mode were used as a reporter of the strength of the bifurcated H bond from a thiol and hydroxyl H(+) at residue i + 4. DFT calculations yielded very similar frequency shifts and an energy of -2.6 and -3.4 kcal/mol for the thiol and hydroxyl bifurcated H bonds, respectively. The strength of the intrahelical bifurcated H bond is consistent with its prevalence in hydrophobic environments and is shown to significantly impact side-chain rotamer distribution.

FTIR spectroscopy has long been used as a tool used to gain average structural information on proteins. With the advent of stable isotope editing, FTIR can be used to derive accurate information on isolated amino acids. In particular, in an anisotropic sample such as membrane layers, it is possible to measure the orientation of the peptidic carbonyl groups. Herein, we review the theory that enables one to obtain accurate restraints from FTIR spectroscopy, alongside considerations for sample suitability and general applicability. We also propose approaches that may be used to generate structural models of simple membrane proteins based on FTIR orientational restraints. This article is part of a Special Issue entitled: FTIR in membrane proteins and peptide studies., (Copyright © 2012. Published by Elsevier B.V.)

Motivation: Most integral membrane proteins form dimeric or oligomeric complexes. Oligomerization is frequently supported by the non-covalent interaction of transmembrane helices. It is currently not clear how many high-affinity transmembrane domains (TMD) exist in a proteome and how specific their interactions are with respect to preferred contacting faces and their underlying residue motifs., Results: We first identify a threshold of 55% sequence similarity, which demarcates the border between meaningful alignments of TMDs and chance alignments. Clustering the human single-span membrane proteome using this threshold groups ~40% of the TMDs. The homotypic interaction of the TMDs representing the 33 largest clusters was systematically investigated under standardized conditions. The results reveal a broad distribution of relative affinities. High relative affinity frequently coincides with (i) the existence of a preferred helix-helix interface and (ii) sequence specificity as indicated by reduced affinity after mutating conserved residues., Supplementary Information: Supplementary data are available at Bioinformatics online.

The polarity pattern of a macromolecule is of utmost importance to its structure and function. For example, one of the main driving forces for protein folding is the burial of hydrophobic residues. Yet polarity remains a difficult property to measure experimentally, due in part to its non-uniformity in the protein interior. Herein, we show that FTIR linewidth analysis of noninvasive 1-(13)C=(18)O labels can be used to obtain a reliable measure of the local polarity, even in a highly multi-phasic system, such as a membrane protein. We show that in the Influenza M2 H(+) channel, residues that line the pore are located in an environment that is as polar as fully solvated residues, while residues that face the lipid acyl chains are located in an apolar environment. Taken together, FTIR linewidth analysis is a powerful, yet chemically non-perturbing approach to examine one of the most important properties in proteins - polarity.

Sodium proton antiporters are ubiquitous membrane proteins that catalyze the exchange of Na(+) for protons throughout the biological world. The Escherichia coli NhaA is the archetypal Na(+)/H(+) antiporter and is absolutely essential for survival in high salt concentrations under alkaline conditions. Its crystal structure, accompanied by extensive molecular dynamics simulations, have provided an atomically detailed model of its mechanism. In this study, we utilized a combination of computational methodologies in order to construct a structural model for the Na(+)/H(+) antiporter from the gram-negative bacterium Vibrio parahaemolyticus. We explored its overall architecture by computational means and validated its stability and robustness. This protein belongs to a novel group of NhaA proteins that transports not only Na(+) and Li(+) as substrate ions, but K(+) as well, and was also found to miss a β-hairpin segment prevalent in other homologs of the Bacteria domain. We propose, for the first time, a structure of a prototype model of a β-hairpin-less NhaA that is selective to K(+). Better understanding of the Vibrio parahaemolyticus NhaA structure-function may assist in studies on ion transport, pH regulation and designing selective blockers.

The interactions between channels and their cognate blockers are at the heart of numerous biomedical phenomena. Herein, we unravel one particularly important example bearing direct pharmaceutical relevance: the blockage mechanism of the influenza M2 channel by the anti-flu amino-adamantyls (amantadine and rimantadine) and how the channel and, consequently, the virus develop resistance against them. Using both computational analyses and experimental verification, we find that amino-adamantyls inhibit M2's H(+) channel activity by electrostatic hindrance due to their positively charged amino group. In contrast, the hydrophobic adamantyl moiety on its own does not impact conductivity. Additionally, we were able to uncover how mutations in M2 are capable of retaining drug binding on the one hand yet rendering the protein and the mutated virus resistant to amino-adamantyls on the other hand. We show that the mutated, drug-resistant protein has a larger binding pocket for the drug. Hence, despite binding the channel, the drug remains sufficiently mobile so as not to exert a H(+)-blocking positive electrostatic hindrance. Such insight into the blocking mechanism of amino-adamantyls, and resistance thereof, may aid in the design of next-generation anti-flu agents.

The recent outbreaks of avian flu in Southeast Asia and swine flu in Mexico City painfully exemplify the ability of the influenza virus to rapidly mutate and develop resistance to modern medicines. This review seeks to detail the molecular mechanism by which the influenza virus has obtained resistance to amino-adamantyls, one of only two classes of drugs that combat the flu. Amino-adamantyls target the viral M2 H(+) channel and have become largely ineffective due to mutations in the transmembrane domain of the protein. Herein we describe these resistance rendering mutations and the compounded effects they have upon the protein's function and resulting virus viability., (2010 Elsevier B.V. All rights reserved.)

The ability to discriminate between highly similar substrates is one of the remarkable properties of enzymes. For example, transporters and channels that selectively distinguish between various solutes enable living organisms to maintain and control their internal environment in the face of a constantly changing surrounding. Herein, we examine in detail the selectivity properties of one of the most important salt transporters: the bacterial Na+/H+ antiporter. Selectivity can be achieved at either the substrate binding step or in subsequent antiporting. Surprisingly, using both computational and experimental analyses synergistically, we show that binding per se is not a sufficient determinant of selectively. All alkali ions from Li+ to Cs+ were able to competitively bind the antiporter's binding site, whether the protein was capable of pumping them or not. Hence, we propose that NhaA's binding site is relatively promiscuous and that the selectivity is determined at a later stage of the transport cycle.

Yersinia pestis, the bacterium that historically accounts for the Black Death epidemics, has nowadays gained new attention as a possible biological warfare agent. In this study, its Na⁺/H⁺ antiporter is investigated for the first time, by a combination of experimental and computational methodologies. We determined the protein's substrate specificity and pH dependence by fluorescence measurements in everted membrane vesicles. Subsequently, we constructed a model of the protein's structure and validated the model using molecular dynamics simulations. Taken together, better understanding of the Yersinia pestis Na⁺/H⁺ antiporter's structure-function relationship may assist in studies on ion transport, mechanism of action and designing specific blockers of Na⁺/H⁺ antiporter to help in fighting Yersinia pestis -associated infections. We hope that our model will prove useful both from mechanistic and pharmaceutical perspectives.

The influenza M2 H(+) channel enables the concomitant acidification of the viral lumen upon endosomic internalization. This process is critical to the viral infectivity cycle, demonstrated by the fact that M2 is one of only two targets for anti-flu agents. However, aminoadamantyls that block the M2 channel are of limited therapeutic use due to the emergence of resistance mutations in the protein. Herein, using an assay that involves expression of the protein in Escherichia coli with resultant growth retardation, we present quantitative measurements of channel blocker interactions. Comparison of detailed K(s) measurements of different drugs for several influenza channels, shows that the swine flu M2 exhibits the highest resistance to aminoadamantyls of any channel known to date. From the perspective of the blocker, we show that rimantadine is consistently a better blocker of M2 than amantadine. Taken together, such detailed and quantitative analyses provide insight into the mechanism of this important and pharmaceutically relevant channel blocker system., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The pH activated M2 H(+) channel from influenza A has been a subject of numerous studies due to following: (1) It serves as a target for the aminoadamantane drugs that block its channel activity. (2) M2's small size makes it amenable to biophysical scrutiny. (3) A single histidine residue is thought to control the pH gating of the channel. Recent FTIR analysis proposed that the helices of the channel rotate about their directors during pH activation. Herein, we report on molecular dynamics simulations of the X-ray structure of the protein with three charged histidine residues, representing the open form of the protein and two rotated forms with neutral histidines, representing its closed form. We compare the channel stability, convergence, interaction with water and hydration of the histidine residues that have been implicated in channel gating. Taken together, we show that both forms of the protein are stable during the course of the MD simulation and that indeed a rotation of the helices leads to channel closure. Finally, we propose a mechanism for channel gating that involves protonation of the histidine residues that necessities their increased solvation.

We used computational methods to study the interaction between two key proteins in apoptosis regulation: the transcription factor NF-kappa-B (NFkappaB) and the proapoptotic protein ASPP2. The C-terminus of ASPP2 contains ankyrin repeats and SH3 domains (ASPP2(ANK-SH3)) that mediate interactions with numerous apoptosis-related proteins, including the p65 subunit of NFkappaB (NFkappaB(p65)). Using peptide-based methods, we have recently identified the interaction sites between NFkappaB(p65) and ASPP2(ANK-SH3) (Rotem et al., J Biol Chem 283, 18990-18999). Here we conducted a computational study of protein docking and molecular dynamics to obtain a structural model of the complex between the full length proteins and propose a mechanism for the interaction. We found that ASPP2(ANK-SH3) binds two sites in NFkappaB(p65), at residues 236-253 and 293-313 that contain the nuclear localization signal (NLS). These sites also mediate the binding of NFkappaB to its natural inhibitor IkappaB, which also contains ankyrin repeats. Alignment of the ankyrin repeats of ASPP2(ANK-SH3) and IkappaB revealed that both proteins share highly similar interfaces at their binding sites to NFkappaB. Protein docking of ASPP2(ANK-SH3) and NFkappaB(p65), as well as molecular dynamics simulations of the proteins, provided structural models of the complex that are energetically similar to the NFkappaB-IkappaB determined structure. Our results show that ASPP2(ANK-SH3) binds NFkappaB(p65) in a similar manner to its natural inhibitor IkappaB, suggesting a possible novel role for ASPP2 as an NFkappaB inhibitor., (2009 Wiley-Liss, Inc.)

The M2 protein is an essential component of the Influenza virus' infectivity cycle. It is a homo-tetrameric bundle forming a pH-gated H(+) channel. The structure of M2 was solved by three different groups, using different techniques, protein sequences and pH environment. For example, solid-state NMR spectroscopy was used on a protein in lipid bilayers, while X-ray crystallography and solution NMR spectroscopy were applied on a protein in detergent micelles. The resulting structures from the above efforts are rather distinct. Herein, we examine the different structures under uniform conditions such as a lipid bilayer and specified protonation state. We employ extensive molecular dynamics simulations, in several protonation states, representing both closed and open forms of the channel. Exploring the properties of each of these structures has shown that the X-ray structure is more stable than the other structures according to various criteria, although its water conductance and water-wire formation do not correlate to the protonation state of the channel.

Within 1 or 2 decades, the reputation of membrane-spanning alpha-helices has changed dramatically. Once mostly regarded as dull membrane anchors, transmembrane domains are now recognized as major instigators of protein-protein interaction. These interactions may be of exquisite specificity in mediating assembly of stable membrane protein complexes from cognate subunits. Further, they can be reversible and regulatable by external factors to allow for dynamic changes of protein conformation in biological function. Finally, these helices are increasingly regarded as dynamic domains. These domains can move relative to each other in different functional protein conformations. In addition, small-scale backbone fluctuations may affect their function and their impact on surrounding lipid shells. Elucidating the ways by which these intricate structural features are encoded by the amino acid sequences will be a fascinating subject of research for years to come.

The pH-controlled M2 protein from influenza A is a critical component of the virus and serves as a target for the aminoadamantane antiflu agents that block its H+ channel activity. To better understand its H+ gating mechanism, we investigated M2 in lipid bilayers with a new combination of IR spectroscopies and theory. Linear Fourier transform infrared (FTIR) spectroscopy was used to measure the precise orientation of the backbone carbonyl groups, and 2D infrared (IR) spectroscopy was used to identify channel-lining residues. At low pH (open state), our results match previously published solid-state NMR and X-ray structures remarkably well. However, at neutral pH when the channel is closed, our measurements indicate that a large conformational change occurs that is consistent with the transmembrane alpha-helices rotating by one amino acid register--a structural rearrangement not previously observed. The combination of simulations and isotope-labeled FTIR and 2D IR spectroscopies provides a noninvasive means of interrogating the structures of membrane proteins in general and ion channels in particular.

Aquaporin 0 (AQP0), the most abundant membrane protein in mammalian lens fiber cells, not only serves as the primary water channel in this tissue but also appears to mediate the formation of thin junctions between fiber cells. AQP0 is remarkably less water permeable than other aquaporins, but the structural basis and biological significance of this low permeability remain uncertain, as does the permeability of the protein in a reported junctional form. To address these issues, we performed molecular dynamics (MD) simulations of water transport through membrane-embedded AQP0 in both its (octameric) junctional and (tetrameric) nonjunctional forms. From our simulations, we measured an osmotic permeability for the nonjunctional form that agrees with experiment and found that the distinct dynamics of the conserved, lumen-protruding side chains of Tyr-23 and Tyr-149 modulate water passage, accounting for the slow permeation. The junctional and nonjunctional forms conducted water equivalently, in contrast to a previous suggestion based on static crystal structures that water conduction is lost on junction formation. Our analysis suggests that the low water permeability of AQP0 may help maintain the mechanical stability of the junction. We hypothesize that the structural features leading to low permeability may have evolved in part to allow AQP0 to form junctions that both conduct water and contribute to the organizational structure of the fiber cell tissue and microcirculation within it, as required to maintain transparency of the lens.

A molecular-level understanding of the function of a protein requires knowledge of both its structural and dynamic properties. NMR spectroscopy allows the measurement of generalized order parameters that provide an atomistic description of picosecond and nanosecond fluctuations in protein structure. Molecular dynamics (MD) simulation provides a complementary approach to the study of protein dynamics on similar time scales. Comparisons between NMR spectroscopy and MD simulations can be used to interpret experimental results and to improve the quality of simulation-related force fields and integration methods. However, apparent systematic discrepancies between order parameters extracted from simulations and experiments are common, particularly for elements of noncanonical secondary structure. In this paper, results from a 1.2 micros explicit solvent MD simulation of the protein ubiquitin are compared with previously determined backbone order parameters derived from NMR relaxation experiments [Tjandra, N.; Feller, S. E.; Pastor, R. W.; Bax, A. J. Am. Chem. Soc. 1995, 117, 12562-12566]. The simulation reveals fluctuations in three loop regions that occur on time scales comparable to or longer than that of the overall rotational diffusion of ubiquitin and whose effects would not be apparent in experimentally derived order parameters. A coupled analysis of internal and overall motion yields simulated order parameters substantially closer to the experimentally determined values than is the case for a conventional analysis of internal motion alone. Improved agreement between simulation and experiment also is encouraging from the viewpoint of assessing the accuracy of long MD simulations.

Na+/H+ antiporters are central to cellular salt and pH homeostasis. The structure of Escherichia coli NhaA was recently determined, but its mechanisms of transport and pH regulation remain elusive. We performed molecular dynamics simulations of NhaA that, with existing experimental data, enabled us to propose an atomically detailed model of antiporter function. Three conserved aspartates are key to our proposed mechanism: Asp164 (D164) is the Na+-binding site, D163 controls the alternating accessibility of this binding site to the cytoplasm or periplasm, and D133 is crucial for pH regulation. Consistent with experimental stoichiometry, two protons are required to transport a single Na+ ion: D163 protonates to reveal the Na+-binding site to the periplasm, and subsequent protonation of D164 releases Na+. Additional mutagenesis experiments further validated the model.

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices from bitopic membrane proteins was analyzed using a relative conservation analysis. Interestingly, the transmembrane domains of bitopic proteins are on average, significantly more conserved than the remainder of the protein, even when taking into account their smaller amino acid repertoire. Analysis of highly conserved transmembrane domains did not reveal any unifying consensus, pointing to a great diversity in their conservation patterns. However, Fourier power spectrum analysis was able to show that regardless of the conservation motif, in most sequences a significant conservation moment was observed, in that one side of the helix was conserved while the other was not. Taken together, it may be possible to conclude that a significant proportion of transmembrane helices from bitopic membrane proteins participate in specific interactions, in a variety of modes in the plane of the lipid bilayer.

In a recently reported study [Mukherjee, et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3528] we used 2D IR spectroscopy and 1-(13)C=(18)O isotope labeling to measure the vibrational dynamics of 11 amide I modes in the CD3zeta transmembrane domain. We found that the homogeneous line widths and population relaxation times were all nearly identical, but that the amount of inhomogeneous broadening correlated with the position of the amide group inside the membrane. In this study, we use molecular dynamics simulations to investigate the structural and dynamical origins of these experimental observations. We use two models to convert the simulations to frequency trajectories from which the mean frequencies, standard deviations, frequency correlation functions, and 2D IR spectra are calculated. Model 1 correlates the hydrogen-bond length to the amide I frequency, whereas model 2 uses an ab initio-based electrostatic model. We find that the structural distributions of the peptidic groups and their environment are reflected in the vibrational dynamics of the amide I modes. Environmental forces from the water and lipid headgroups partially denature the helices, shifting the infrared frequencies and creating larger inhomogeneous distributions for residues near the ends. The least inhomogeneously broadened residues are those located in the middle of the membrane where environmental electrostatic forces are weakest and the helices are most ordered. Comparison of the simulations to experiment confirms that the amide I modes near the C-terminal are larger than at the N-terminal because of the asymmetric structure of the peptide bundle in the membrane. The comparison also reveals that residues at a kink in the alpha-helices have broader line widths than more helical parts of the peptide because the peptide backbone at the kink exhibits a larger amount of structural disorder. Taken together, the simulations and experiments reveal that infrared line shapes are sensitive probes of membrane protein structural and environmental heterogeneity.

We have investigated the effect of the transmembrane domain of three viral ion channel proteins on the lipid bilayer structure by X-ray reflectivity and scattering from oriented planar bilayers. The proteins show a similar effect on the lipid bilayer structural parameters: an increase in the lipid bilayer hydrophobic core, a decrease in the amplitude of the vertical density profile and a systematic change in the ordering of the acyl chains as a function of protein-to-lipid ratio. These results are discussed in a comparative view.

Fourier transform infrared (FTIR) spectroscopy has long been a powerful tool for structural analysis of membrane proteins. However, because of difficulties in resolving contributions from individual residues, most of the derived measurements tend to yield average properties for the system under study. Isotope editing, through its ability to resolve individual vibrations, establishes FTIR as a method that is capable of yielding accurate structural data on individual sites in a protein.