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Roy Duncan - One of the best experts on this subject based on the ideXlab platform.

  • liposome reconstitution of a minimal Protein mediated Membrane Fusion machine
    The EMBO Journal, 2005
    Co-Authors: Deniz Top, Jennifer A. Corcoran, Jayme Salsman, Roberto De Antueno, Jamie S Mader, David W Hoskin, Ahmed Touhami, M H Jericho, Roy Duncan
    Abstract:

    Biological Membrane Fusion is dependent on Protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of Protein-mediated Membrane Fusion involves the sequential refolding of complex homomeric or heteromeric Protein Fusion machines. The structural features of a new family of Fusion-associated small transMembrane (FAST) Proteins appear incompatible with existing models of Membrane Fusion Protein function. While the FAST Proteins function to induce efficient cell–cell Fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate Membrane Fusion or are dependent on cellular Protein cofactors. Using proteoliposomes containing the purified p14 FAST Protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome Fusion assays that p14 is both necessary and sufficient for Membrane Fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated Membrane Fusion rivals that of the more complex cellular and viral Fusion Proteins, making the FAST Proteins the simplest known Membrane Fusion machines.

  • Unusual Topological Arrangement of Structural Motifs in the Baboon Reovirus Fusion-Associated Small TransMembrane Protein
    Journal of virology, 2005
    Co-Authors: Sandra Dawe, Jennifer A. Corcoran, Jayme Salsman, Eileen K. Clancy, Roy Duncan
    Abstract:

    Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell Fusion through a distinct class of Membrane Fusion-inducing Proteins referred to as the Fusion-associated small transMembrane (FAST) Proteins. The p15 Membrane Fusion Protein of baboon reovirus is unique among the FAST Proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transMembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is Membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 Membrane insertion and a bitopic N(exoplasmic)/C(cytoplasmic) topology. This topology results in the translocation of the smallest ectodomain ( approximately 20 residues) of any known viral Fusion Protein, with the majority of p15 positioned on the cytosolic side of the Membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the Fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a Fusion peptide motif, a hallmark feature of Membrane Fusion Proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus Membrane Fusion Protein necessitate alternate models of Protein-mediated Membrane Fusion.

  • Cell-Cell Fusion Induced by the Avian Reovirus Membrane Fusion Protein Is Regulated by Protein Degradation
    Journal of virology, 2004
    Co-Authors: Maya. Shmulevitz, Jennifer A. Corcoran, Jayme Salsman, Roy Duncan
    Abstract:

    The p10 Fusion-associated small transMembrane Protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell Fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the Protein for degradation following p10 insertion into an early Membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate Fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell Fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral Membrane Fusion Protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell Fusion.

  • Reptilian Reovirus Utilizes a Small Type III Protein with an External Myristylated Amino Terminus To Mediate Cell-Cell Fusion
    Journal of virology, 2004
    Co-Authors: Jennifer A. Corcoran, Roy Duncan
    Abstract:

    Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell Fusion. A small, hydrophobic, basic, 125-amino-acid Fusion Protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this Fusion activity. Sequence comparisons to previously characterized reovirus Fusion Proteins indicated that p14 represents a new member of the Fusion-associated small transMembrane (FAST) Protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral Membrane Proteins, the type III Proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct Membrane insertion and Protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell Membrane. The topology and structural motifs present in this novel reovirus Membrane Fusion Protein further accentuate the diversity and unusual properties of the FAST Protein family and clearly indicate that the FAST Proteins represent a third distinct class of viral Membrane Fusion Proteins.

  • Structural and Functional Properties of an Unusual Internal Fusion Peptide in a Nonenveloped Virus Membrane Fusion Protein
    Journal of virology, 2004
    Co-Authors: Maya. Shmulevitz, Raquel F. Epand, Roy Duncan
    Abstract:

    The avian and Nelson Bay reoviruses are two of only a limited number of nonenveloped viruses capable of inducing cell-cell Membrane Fusion. These viruses encode the smallest known Membrane Fusion Proteins (p10). We now show that a region of moderate hydrophobicity we call the hydrophobic patch (HP), present in the small N-terminal ectodomain of p10, shares the following characteristics with the Fusion peptides of enveloped virus Fusion Proteins: (i) an abundance of glycine and alanine residues, (ii) a potential amphipathic secondary structure, (iii) Membrane-seeking characteristics that correspond to the degree of hydrophobicity, and (iv) the ability to induce lipid mixing in a liposome Fusion assay. The p10 HP is therefore predicted to provide a function in the mechanism of Membrane Fusion similar to those of the Fusion peptides of enveloped virus Fusion peptides, namely, association with and destabilization of opposing lipid bilayers. Mutational and biophysical analysis suggested that the internal Fusion peptide of p10 lacks alpha-helical content and exists as a disulfide-stabilized loop structure. Similar kinked structures have been reported in the Fusion peptides of several enveloped virus Fusion Proteins. The preservation of a predicted loop structure in the Fusion peptide of this unusual nonenveloped virus Membrane Fusion Protein supports an imperative role for a kinked Fusion peptide motif in biological Membrane Fusion.

Taiji Nakae - One of the best experts on this subject based on the ideXlab platform.

  • Assignment of the outer-Membrane-subunit-selective domain of the Membrane Fusion Protein in the tripartite xenobiotic efflux pump of Pseudomonas aeruginosa.
    FEMS microbiology letters, 2006
    Co-Authors: Shima Eda, Hideaki Maseda, Eisaku Yoshihara, Taiji Nakae
    Abstract:

    Early in vivo experiments revealed that the MexA-MexB dipartite pump unit of Pseudomonas aeruginosa conferred drug resistance to the cells, which expressed OprM, but not to the OprN-bearing cells. While the MexE-MexF unit interplayed with either the outer Membrane subunits. Taking advantage of this subunit selectivity, we selected the MexA mutant that gained the ability to interplay with OprN. Four mutants have been isolated and all showed an amino acid substitution (Q116R) in the coiled-coil domain of MexA. The hybrid Protein bearing the coiled-coil domain of MexA and the remainder domains from MexE retained the ability to interplay with OprM, but lost the functional interplay with OprN. These results established that the coiled-coil domain of the Membrane Fusion Protein is responsible for selecting the compatible outer Membrane subunit.

  • Role of the Membrane Fusion Protein in the assembly of resistance-nodulation-cell division multidrug efflux pump in Pseudomonas aeruginosa.
    Biochemical and biophysical research communications, 2004
    Co-Authors: Vladislav V. Mokhonov, Ekaterina I. Mokhonova, Hiroyuki Akama, Taiji Nakae
    Abstract:

    The tripartite xenobiotic-antibiotic transporter of Pseudomonas aeruginosa consists of the inner Membrane transporter (e.g., MexB, MexY), the periplasmic Membrane-Fusion-Protein (e.g., MexA, MexX), and the outer Membrane channel Protein (e.g., OprM). These subunits were assumed to assemble into a transporter unit during export of the substrates. However, subunit interaction and their specificity in native form remained to be elucidated. To address these important questions, we analyzed the role of the individual subunits for the assembly of MexAB-OprM by pull-down assay tagging only one of the subunits. We found stable MexA-MexB-OprM complex without chemical cross-linking that withstand all purification procedures. Results of bi-partite interactions analysis showed tight association between MexA and OprM in the absence of MexB, whereas the expression systems lacking MexA failed to co-purify MexB or OprM. None of the heterologous subunit combinations such as MexA+MexY(his)+OprM and MexX+MexB(his)+OprM showed interaction. These results implied that the Membrane Fusion Protein is central to the tripartite xenobiotic transporter assembly.

  • crystal structure of the Membrane Fusion Protein mexa of the multidrug transporter in pseudomonas aeruginosa
    Journal of Biological Chemistry, 2004
    Co-Authors: Hiroyuki Akama, Takanori Matsuura, Sachiko Kashiwagi, Hiroshi Yoneyama, Shin-ichiro Narita, Tomitake Tsukihara, Atsushi Nakagawa, Taiji Nakae
    Abstract:

    The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the Membrane Fusion Protein, the body of the transporter, and the outer Membrane channel Protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 A. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new Protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod α-helical domain was globular and constructed by a cluster of eight short β-sheets. The third domain located distal to the α-helical rod was globular and composed of seven short β-sheets and one short α-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic α-barrel domain of OprM via the α-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the Membrane bridge Protein.

  • Crystal Structure of the Membrane Fusion Protein, MexA, of the Multidrug Transporter in Pseudomonas aeruginosa
    The Journal of biological chemistry, 2004
    Co-Authors: Hiroyuki Akama, Takanori Matsuura, Sachiko Kashiwagi, Hiroshi Yoneyama, Shin-ichiro Narita, Tomitake Tsukihara, Atsushi Nakagawa, Taiji Nakae
    Abstract:

    The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the Membrane Fusion Protein, the body of the transporter, and the outer Membrane channel Protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 A. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new Protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod alpha-helical domain was globular and constructed by a cluster of eight short beta-sheets. The third domain located distal to the alpha-helical rod was globular and composed of seven short beta-sheets and one short alpha-helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic alpha-barrel domain of OprM via the alpha-helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the Membrane bridge Protein.

  • Function of the Membrane Fusion Protein, MexA, of the MexA, B-OprM Efflux Pump in Pseudomonas aeruginosa without an Anchoring Membrane
    The Journal of biological chemistry, 2000
    Co-Authors: Hiroshi Yoneyama, Hideaki Maseda, Hiroshi Kamiguchi, Taiji Nakae
    Abstract:

    Resistance of Pseudomonas aeruginosa to multiple species of antibiotics is largely attributable to expression of the MexA, B-OprM efflux pump. The MexA Protein is thought to be located at the inner Membrane and has been assumed to link the xenobiotics-exporting subunit, MexB, and the outer Membrane channel Protein, OprM. To verify this assumption, we analyzed Membrane anchoring and localization of the MexA Protein. n-[9, 10-(3)H]Palmitic acid incorporation experiments revealed that MexA was radiolabeled with palmitic acid, suggesting that the MexA anchors the inner Membrane via the fatty acid moiety. To evaluate the role of lipid modification and inner Membrane anchoring, we substituted cysteine 24 with phenylalanine or tyrosine and tested whether or not these mutant MexAs function properly. When the mutant mexAs were expressed in the strain lacking chromosomal mexA in the presence of n-[9,10-(3)H]palmitic acid, we found undetectable radiolabeling at the MexA band. These transformants restored antibiotic resistance to the level of the wild-type strain, indicating that lipid modification is not essential for MexA function. These mutant strains contained both processed and unprocessed forms of the MexA Proteins. Cellular fractionation experiments revealed that an unprocessed form of MexA anchored the inner Membrane probably via an uncleaved signal sequence, whereas the processed form was undetectable in the Membrane fraction. To assure that the lipid-free MexA polypeptide could be unbound to the Membrane, we analyzed the two-dimensional Membrane topology by the gene Fusion technique. A total of 78 mexA-blaM Fusions covering the entire MexA polypeptide were constructed, and all Fusion sites were shown to be located at the periplasm. To answer the question of whether or not Membrane anchoring is essential for the MexA function, we replaced the signal sequence of the MexA Protein with that of the azurin Protein, which contains a cleavable signal sequence but no lipid modification site. The signal sequence of the azurin-MexA hybrid Protein was properly processed and bore the mature MexA, which was fully recovered in the soluble fraction. The transformant, which expressed azurin-MexA hybrid Protein restored the antibiotic resistance to a level indistinguishable from that of the wild-type strain. We concluded from these results that the MexA Protein is fully functional as expressed in the periplasmic space without anchoring the inner Membrane. This finding questioned the assumption that the Membrane Fusion Proteins connect the inner and outer Membranes.

Helen I. Zgurskaya - One of the best experts on this subject based on the ideXlab platform.

  • MacA, a periplasmic Membrane Fusion Protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity
    Journal of bacteriology, 2013
    Co-Authors: Helen I. Zgurskaya
    Abstract:

    The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic Membrane Fusion Protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA Protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.

  • Multicomponent drug efflux complexes: architecture and mechanism of assembly
    Future microbiology, 2009
    Co-Authors: Helen I. Zgurskaya
    Abstract:

    Multidrug efflux pumps are major contributors to intrinsic antibiotic resistance in Gram-negative pathogens. The basic structure of these pumps comprises an inner Membrane transporter, a periplasmic Membrane Fusion Protein and an outer Membrane channel. However, the architecture and composition of multidrug efflux complexes vary significantly because of the topological and functional diversity of the inner Membrane transporters. This article presents the current views on architecture and assembly of multicomponent drug efflux transporters from Gram-negative bacteria.

  • Fitting Periplasmic Membrane Fusion Proteins to Inner Membrane Transporters: Mutations That Enable Escherichia coli AcrA To Function with Pseudomonas aeruginosa MexB
    Journal of bacteriology, 2007
    Co-Authors: Ganesh Krishnamoorthy, Elena B. Tikhonova, Helen I. Zgurskaya
    Abstract:

    AcrAB-TolC from Escherichia coli is a multidrug efflux complex capable of transenvelope transport. In this complex, AcrA is a periplasmic Membrane Fusion Protein that establishes a functional connection between the inner Membrane transporter AcrB of the RND superfamily and the outer Membrane channel TolC. To gain insight into the mechanism of the functional association between components of this complex, we replaced AcrB with its close homolog MexB from Pseudomonas aeruginosa. Surprisingly, we found that AcrA is promiscuous and can form a partially functional complex with MexB and TolC. The chimeric AcrA-MexB-TolC complex protected cells from sodium dodecyl sulfate, novobiocin, and ethidium bromide but failed with other known substrates of MexB. We next identified single and double mutations in AcrA and MexB that enabled the complete functional fit between AcrA, MexB, and TolC. Mutations in either the alpha-helical hairpin of AcrA making contact with TolC or the beta-barrel domain lying on MexB improved the functional alignment between components of the complex. Our results suggest that three components of multidrug efflux pumps do not associate in an "all-or-nothing" fashion but accommodate a certain degree of flexibility. This flexibility in the association between components affects the transport efficiency of RND pumps.

  • Reconstitution of the Escherichia coli macrolide transporter: the periplasmic Membrane Fusion Protein MacA stimulates the ATPase activity of MacB.
    Molecular microbiology, 2007
    Co-Authors: Elena B. Tikhonova, Vishakha K. Devroy, Sze Yi Lau, Helen I. Zgurskaya
    Abstract:

    Periplasmic Membrane Fusion Proteins (MFPs) are essential components of the type I Protein secretion systems and drug efflux pumps in Gram-negative bacteria. Previous studies suggested that MFPs connect the inner and outer Membrane components of the transport systems and by this means co-ordinate the transfer of substrates across the two Membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC-type transporter. Similar to other MFP-dependent transporters from E. coli, the in vivo function of MacAB requires the outer Membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis-Menten kinetics but was unresponsive to substrates or accessory Proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter.

  • pH-induced Conformational Changes of AcrA, the Membrane Fusion Protein of Escherichia coli Multidrug Efflux System
    The Journal of biological chemistry, 2003
    Co-Authors: Kelly G. Stratton, Helen I. Zgurskaya, Jun Liu
    Abstract:

    The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer Membrane Proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral Membrane Fusion Proteins, and that AcrA actively mediates the efflux of substrates.

Margaret Kielian - One of the best experts on this subject based on the ideXlab platform.

  • The domain I-domain III linker plays an important role in the fusogenic conformational change of the alphavirus Membrane Fusion Protein.
    Journal of virology, 2011
    Co-Authors: Yan Zheng, Claudia Sánchez-san Martín, Zhao-ling Qin, Margaret Kielian
    Abstract:

    The alphavirus Semliki Forest virus (SFV) infects cells through a low-pH-dependent Membrane Fusion reaction mediated by the virus Fusion Protein E1. Acidic pH initiates a series of E1 conformational changes that culminate in Membrane Fusion and include dissociation of the E1/E2 heterodimer, insertion of the E1 Fusion loop into the target Membrane, and refolding of E1 to a stable trimeric hairpin conformation. A highly conserved histidine (H3) on the E1 Protein was previously shown to promote low-pH-dependent E1 refolding. An SFV mutant with an alanine substitution at this position (H3A) has a lower pH threshold and reduced efficiency of virus Fusion and E1 trimer formation than wild-type SFV. Here we addressed the mechanism by which H3 promotes E1 refolding and Membrane Fusion. We identified E1 mutations that rescue the H3A defect. These revertants implicated a network of interactions that connect the domain I-domain III (DI-DIII) linker region with the E1 core trimer, including H3. In support of the importance of these interactions, mutation of residues in the network resulted in more acidic pH thresholds and reduced efficiencies of Membrane Fusion. In vitro studies of truncated E1 Proteins demonstrated that the DI-DIII linker was required for production of a stable E1 core trimer on target Membranes. Together, our results suggest a critical and previously unidentified role for the DI-DIII linker region during the low-pH-dependent refolding of E1 that drives Membrane Fusion.

  • Alphavirus Entry and Membrane Fusion.
    Viruses, 2010
    Co-Authors: Margaret Kielian, Chantal Chanel-vos, Maofu Liao
    Abstract:

    The study of enveloped animal viruses has greatly advanced our understanding of the general properties of Membrane Fusion and of the specific pathways that viruses use to infect the host cell. The Membrane Fusion Proteins of the alphaviruses and flaviviruses have many similarities in structure and function. As reviewed here, alphaviruses use receptor-mediated endocytic uptake and low pH-triggered Membrane Fusion to deliver their RNA genomes into the cytoplasm. Recent advances in understanding the biochemistry and structure of the alphavirus Membrane Fusion Protein provide a clearer picture of this Fusion reaction, including the Protein’s conformational changes during Fusion and the identification of key domains. These insights into the alphavirus Fusion mechanism suggest new areas for experimental investigation and potential inhibitor strategies for anti-viral therapy.

  • In Vitro Reconstitution Reveals Key Intermediate States of Trimer Formation by the Dengue Virus Membrane Fusion Protein
    Journal of virology, 2010
    Co-Authors: Maofu Liao, Claudia Sánchez-san Martín, Aihua Zheng, Margaret Kielian
    Abstract:

    The flavivirus dengue virus (DV) infects cells through a low-pH-triggered Membrane Fusion reaction mediated by the viral envelope Protein E. E is an elongated transMembrane Protein with three domains and is organized as a homodimer on the mature virus particle. During Fusion, the E Protein homodimer dissociates, inserts the hydrophobic Fusion loop into target Membranes, and refolds into a trimeric hairpin in which domain III (DIII) packs against the central trimer. It is clear that E refolding drives Membrane Fusion, but the steps in hairpin formation and their pH requirements are unclear. Here, we have used truncated forms of the DV E Protein to reconstitute trimerization in vitro. Protein constructs containing domains I and II (DI/II) were monomeric and interacted with Membranes to form core trimers. DI/II-Membrane interaction and trimerization occurred efficiently at both neutral and low pH. The DI/II core trimer was relatively unstable and could be stabilized by binding exogenous DIII or by the formation of mixed trimers containing DI/II plus E Protein with all three domains. The mixed trimer had unoccupied DIII interaction sites that could specifically bind exogenous DIII at either low or neutral pH. Truncated DV E Proteins thus reconstitute hairpin formation and define properties of key domain interactions during DV Fusion.

  • Second-Site Revertants of a Semliki Forest Virus Fusion-Block Mutation Reveal the Dynamics of a Class II Membrane Fusion Protein
    Journal of virology, 2006
    Co-Authors: Chantal Chanel-vos, Margaret Kielian
    Abstract:

    The alphavirus Semliki Forest virus (SFV) infects cells through low-pH-induced Membrane Fusion mediated by the E1 Protein, a class II virus Membrane Fusion Protein. During Fusion, E1 inserts into target Membranes via its hydrophobic Fusion loop and refolds to form a stable E1 homotrimer. Mutation of a highly conserved histidine (the H230A mutation) within a loop adjacent to the Fusion loop was previously shown to block SFV Fusion and infection, although the mutant E1 Protein still inserts into target Membranes and forms a homotrimer. Here we report on second-site mutations in E1 that rescue the H230A mutant. These mutations were located in a cluster within the hinge region, at the Membrane-interacting tip, and within the groove where the E1 stem is believed to pack. Together the revertants reveal specific and interconnected aspects of the Fusion Protein refolding reaction.

Jeremy C. Smith - One of the best experts on this subject based on the ideXlab platform.

  • Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump.
    ACS Infectious Diseases, 2016
    Co-Authors: Narges Abdali, Keith M. Haynes, Julie L. Chaney, Adam T. Green, David Wolloscheck, John K. Walker, Valentin V. Rybenkov, Jerome Baudry, Jerry M Parks, Jeremy C. Smith
    Abstract:

    Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for the development of effective EPIs, especially in light of constantly emerging resistance. Here, we describe EPIs that interact with periplasmic Membrane Fusion Proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-Membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antibiotics in Escherichia coli and other Gram-negative bacteria. Our findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promisi...

  • Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump.
    ACS infectious diseases, 2016
    Co-Authors: Narges Abdali, Keith M. Haynes, Julie L. Chaney, Adam T. Green, David Wolloscheck, John K. Walker, Valentin V. Rybenkov, Jerome Baudry, Jerry M Parks, Jeremy C. Smith
    Abstract:

    Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for the development of effective EPIs, especially in light of constantly emerging resistance. Here, we describe EPIs that interact with periplasmic Membrane Fusion Proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-Membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes, and potentiate the activities of antibiotics in Escherichia coli and other Gram-negative bacteria. Our findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.