[Show abstract][Hide abstract] ABSTRACT: 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 posses 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.
PLoS ONE 10/2014; 9(10):e105387. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 03/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Influenza 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.
Biochimica et Biophysica Acta 09/2013; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 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 transmembrane domains and chance alignments. Clustering the human single-span membrane proteome using this threshold groups ∼40% of the transmembrane domains. The homotypic interaction of the transmembrane domains 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. CONTACT: email@example.com.
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 11/2011; 6(11):e26115. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 10/2011; 6(10):e25182. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of the American Chemical Society 06/2011; 133(25):9903-11. · 11.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochimica et Biophysica Acta 02/2011; 1808(2):547-53. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochimica et Biophysica Acta 01/2011; 1808(1):394-8. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular Modeling 11/2010; 17(8):1877-90. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Proteins Structure Function and Bioinformatics 06/2009; 77(3):602-11. · 3.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Protein Science 06/2009; 18(7):1343-58. · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biophysics of Structure and Mechanism 05/2009; 39(7):1043-9. · 2.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Molecular Modeling 05/2009; 15(11):1317-28. · 1.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.