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BBA - Biomembranes (v.1808, #2)
Introduction to the Special Issue on viral channel forming proteins
by Wolfgang B. Fischer (pp. 509-509).
Introduction to the Special Issue on viral channel forming proteins
by Wolfgang B. Fischer (pp. 509-509).
Viral proteins function as ion channels by Kai Wang; Shiqi Xie; Bing Sun (pp. 510-515).
Viral ion channels are short membrane proteins with 50–120 amino acids and play an important role either in regulating virus replication, such as virus entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. This review summarizes the recent advances in viral encoded ion channel proteins (or viroporins), including PBCV-1 KcV, influenza M2, HIV-1 Vpu, HCV p7, picornavirus 2B, and coronavirus E and 3a. We focus on their function and mechanisms, and also discuss viral ion channel protein serving as a potential drug target.
Keywords: Viral ion channels; PBCV-1; Influenza; HIV-1; HCV; Picornavirus; Coronavirus
Viral proteins function as ion channels by Kai Wang; Shiqi Xie; Bing Sun (pp. 510-515).
Viral ion channels are short membrane proteins with 50–120 amino acids and play an important role either in regulating virus replication, such as virus entry, assembly and release or modulating the electrochemical balance in the subcellular compartments of host cells. This review summarizes the recent advances in viral encoded ion channel proteins (or viroporins), including PBCV-1 KcV, influenza M2, HIV-1 Vpu, HCV p7, picornavirus 2B, and coronavirus E and 3a. We focus on their function and mechanisms, and also discuss viral ion channel protein serving as a potential drug target.
Keywords: Viral ion channels; PBCV-1; Influenza; HIV-1; HCV; Picornavirus; Coronavirus
Functional reconstitution of influenza A M2(22–62) by Emily Peterson; Ted Ryser; Spencer Funk; Daniel Inouye; Mukesh Sharma; Huajun Qin; Timothy A. Cross; David D. Busath (pp. 516-521).
Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K+] gradient-induced membrane potential and [H+] gradient), M2(22–62) showed a transport rate of 78H+/tetramer-s (pHo 6.0, pHi 8.0, nominal Vm=−114mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05–1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K+ flux of 0.1K+/tetramer-s, and also demonstrated that the K+ permeability, relative to H+, was 2.8×10−6. Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H+ selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pKa of 6.
Keywords: M2 protein; Influenza A virus; Liposome assay; Acid activation; Specific transport activity; Membrane protein
Functional reconstitution of influenza A M2(22–62) by Emily Peterson; Ted Ryser; Spencer Funk; Daniel Inouye; Mukesh Sharma; Huajun Qin; Timothy A. Cross; David D. Busath (pp. 516-521).
Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K+] gradient-induced membrane potential and [H+] gradient), M2(22–62) showed a transport rate of 78H+/tetramer-s (pHo 6.0, pHi 8.0, nominal Vm=−114mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05–1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K+ flux of 0.1K+/tetramer-s, and also demonstrated that the K+ permeability, relative to H+, was 2.8×10−6. Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H+ selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pKa of 6.
Keywords: M2 protein; Influenza A virus; Liposome assay; Acid activation; Specific transport activity; Membrane protein
Influenza M2 proton channels by Rafal M. Pielak; James J. Chou (pp. 522-529).
M2 of the influenza virus is an intriguing transmembrane protein that forms a minuscule proton channel in the viral envelope. Its recognized function is to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. It is vital for viral replication and it is a target for the anti-influenza drugs, amantadine and rimantadine. Recently, high resolution structures of M2 channels of both flu A and B have been obtained, providing the desperately needed structural details for understanding the mechanism of proton conductance. In particular, the establishment of the functional solution NMR system of the proton channels enabled simultaneous high resolution structure characterization and measurement of channel dynamics coupled to channel activity. This review summarizes our current understanding of how protons are conducted through the M2 channel from a structural point of view, as well as the modes by which important channel gating elements function during proton conduction.
Keywords: M2; AM2; BM2; Proton channel; Influenza; Flu
Influenza M2 proton channels by Rafal M. Pielak; James J. Chou (pp. 522-529).
M2 of the influenza virus is an intriguing transmembrane protein that forms a minuscule proton channel in the viral envelope. Its recognized function is to equilibrate pH across the viral membrane during cell entry and across the trans-Golgi membrane of infected cells during viral maturation. It is vital for viral replication and it is a target for the anti-influenza drugs, amantadine and rimantadine. Recently, high resolution structures of M2 channels of both flu A and B have been obtained, providing the desperately needed structural details for understanding the mechanism of proton conductance. In particular, the establishment of the functional solution NMR system of the proton channels enabled simultaneous high resolution structure characterization and measurement of channel dynamics coupled to channel activity. This review summarizes our current understanding of how protons are conducted through the M2 channel from a structural point of view, as well as the modes by which important channel gating elements function during proton conduction.
Keywords: M2; AM2; BM2; Proton channel; Influenza; Flu
Computational study of drug binding to the membrane-bound tetrameric M2 peptide bundle from influenza A virus by Ekta Khurana; Russell H. DeVane; Matteo Dal Peraro; Michael L. Klein (pp. 530-537).
The M2 protein of influenza A virus performs the crucial function of transporting protons to the interior of virions enclosed in the endosome. Adamantane drugs, amantadine (AMN) and rimantidine (RMN), block the proton conduction in some strains, and have been used for the treatment and prophylaxis of influenza A infections. The structures of the transmembrane (TM) region of M2 that have been solved in micelles using NMR (residues 23-60) (Schnell and Chou, 2008) and by X-ray crystallography (residues 22-46) (Stouffer et al., 2008) suggest different drug binding sites: external and internal for RMN and AMN, respectively. We have used molecular dynamics (MD) simulations to investigate the nature of the binding site and binding mode of adamantane drugs on the membrane-bound tetrameric M2-TM peptide bundles using as initial conformations the low-pH AMN-bound crystal structure, a high-pH model derived from the drug-free crystal structure, and the high-pH NMR structure. The MD simulations indicate that under both low- and high-pH conditions, AMN is stable inside the tetrameric bundle, spanning the region between residues Val27 to Gly34. At low pH the polar group of AMN is oriented toward the His37 gate, while under high-pH conditions its orientation exhibits large fluctuations. The present MD simulations also suggest that AMN and RMN molecules do not show strong affinity to the external binding sites.
Keywords: Molecular dynamics; Simulations; Amantadine; Adamantane; Transmembrane; Ion channel
Computational study of drug binding to the membrane-bound tetrameric M2 peptide bundle from influenza A virus by Ekta Khurana; Russell H. DeVane; Matteo Dal Peraro; Michael L. Klein (pp. 530-537).
The M2 protein of influenza A virus performs the crucial function of transporting protons to the interior of virions enclosed in the endosome. Adamantane drugs, amantadine (AMN) and rimantidine (RMN), block the proton conduction in some strains, and have been used for the treatment and prophylaxis of influenza A infections. The structures of the transmembrane (TM) region of M2 that have been solved in micelles using NMR (residues 23-60) (Schnell and Chou, 2008) and by X-ray crystallography (residues 22-46) (Stouffer et al., 2008) suggest different drug binding sites: external and internal for RMN and AMN, respectively. We have used molecular dynamics (MD) simulations to investigate the nature of the binding site and binding mode of adamantane drugs on the membrane-bound tetrameric M2-TM peptide bundles using as initial conformations the low-pH AMN-bound crystal structure, a high-pH model derived from the drug-free crystal structure, and the high-pH NMR structure. The MD simulations indicate that under both low- and high-pH conditions, AMN is stable inside the tetrameric bundle, spanning the region between residues Val27 to Gly34. At low pH the polar group of AMN is oriented toward the His37 gate, while under high-pH conditions its orientation exhibits large fluctuations. The present MD simulations also suggest that AMN and RMN molecules do not show strong affinity to the external binding sites.
Keywords: Molecular dynamics; Simulations; Amantadine; Adamantane; Transmembrane; Ion channel
Drug sensitivity, drug-resistant mutations, and structures of three conductance domains of viral porins by Mukesh Sharma; Conggang Li; David D. Busath; Huan-Xiang Zhou; Timothy A. Cross (pp. 538-546).
Recent controversies associated with the structure of the M2 protein from influenza A virus and the binding site of drug molecules amantadine and rimantadine motivated the comparison here of the drug binding to three viral porins including the M2 proteins from influenza A and B as well as the viral protein ‘u’ from HIV-1. While the M2 protein from influenza B does not normally bind amantadine, chimeras with the M2 protein from influenza A show blockage by amantadine. Similarly, Vpu does not normally bind rimantadine, but the single site mutation A18H converts a non-specific channel to a selective proton channel that is sensitive to rimantadine. The comparison of structures and amino acid sequences shows that the membrane protein sample environment can have a significant influence on the structural result. While a bilayer surface bound amphipathic helix has been characterized for AM2, such a helix may be possible for BM2 although it has evaded structural characterization in detergent micelles. A similar amphipathic helix seems less likely for Vpu. Even though the A18H Vpu mutant forms rimantadine sensitive proton channels, the binding of drug and its influence on the protein structure appears to be very different from that for the M2 proteins. Indeed, drug binding and drug resistance in these viral porins appears to result from a complex set of factors.
Keywords: M2 protein; Influenza A virus; Influenza B virus; Viral protein u; HIV; PISEMA; Solid-state NMR; Membrane protein
Drug sensitivity, drug-resistant mutations, and structures of three conductance domains of viral porins by Mukesh Sharma; Conggang Li; David D. Busath; Huan-Xiang Zhou; Timothy A. Cross (pp. 538-546).
Recent controversies associated with the structure of the M2 protein from influenza A virus and the binding site of drug molecules amantadine and rimantadine motivated the comparison here of the drug binding to three viral porins including the M2 proteins from influenza A and B as well as the viral protein ‘u’ from HIV-1. While the M2 protein from influenza B does not normally bind amantadine, chimeras with the M2 protein from influenza A show blockage by amantadine. Similarly, Vpu does not normally bind rimantadine, but the single site mutation A18H converts a non-specific channel to a selective proton channel that is sensitive to rimantadine. The comparison of structures and amino acid sequences shows that the membrane protein sample environment can have a significant influence on the structural result. While a bilayer surface bound amphipathic helix has been characterized for AM2, such a helix may be possible for BM2 although it has evaded structural characterization in detergent micelles. A similar amphipathic helix seems less likely for Vpu. Even though the A18H Vpu mutant forms rimantadine sensitive proton channels, the binding of drug and its influence on the protein structure appears to be very different from that for the M2 proteins. Indeed, drug binding and drug resistance in these viral porins appears to result from a complex set of factors.
Keywords: M2 protein; Influenza A virus; Influenza B virus; Viral protein u; HIV; PISEMA; Solid-state NMR; Membrane protein
Resistance characteristics of influenza to amino-adamantyls by Peleg Astrahan; Isaiah T. Arkin (pp. 547-553).
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.
Keywords: Influenza; Viral resistance; M2 channel; Channel blockers
Resistance characteristics of influenza to amino-adamantyls by Peleg Astrahan; Isaiah T. Arkin (pp. 547-553).
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.
Keywords: Influenza; Viral resistance; M2 channel; Channel blockers
Comparative NMR studies demonstrate profound differences between two viroporins: p7 of HCV and Vpu of HIV-1 by Gabriel A. Cook; Hua Zhang; Sang Ho Park; Yan Wang; Stanley J. Opella (pp. 554-560).
The p7 protein from hepatitis C virus and the Vpu protein from HIV-1 are members of the viroporin family of small viral membrane proteins. It is essential to determine their structures in order to obtain an understanding of their molecular mechanisms and to develop new classes of anti-viral drugs. Because they are membrane proteins, it is challenging to study them in their native phospholipid bilayer environments by most experimental methods. Here we describe applications of NMR spectroscopy to both p7 and Vpu. Isotopically labeled p7 and Vpu samples were prepared by heterologous expression in bacteria, initial isolation as fusion proteins, and final purification by chromatography. The purified proteins were studied in the model membrane environments of micelles by solution NMR spectroscopy and in aligned phospholipid bilayers by solid-state NMR spectroscopy. The resulting structural findings enable comparisons to be made between the two proteins, demonstrating that they have quite different architectures. Most notably, Vpu has one trans-membrane helix and p7 has two trans-membrane helices; in addition, there are significant differences in the structures and dynamics of their internal loop and terminal regions.► It is feasible to obtain high resolution NMR spectra of p7 and Vpu in both micelle and bilayer environments. ► Both p7 and Vpu have complex architectures with both highly structured and mobile regions. ► p7 and Vpu have different architectures, since p7 has two trans-membrane helices and Vpu has one trans-membrane helix. There are other differences as well.
Keywords: Hepatitis C virus; p7; Membrane protein; Vpu; Human immunodeficiency virus; Nuclear magnetic resonance
Comparative NMR studies demonstrate profound differences between two viroporins: p7 of HCV and Vpu of HIV-1 by Gabriel A. Cook; Hua Zhang; Sang Ho Park; Yan Wang; Stanley J. Opella (pp. 554-560).
The p7 protein from hepatitis C virus and the Vpu protein from HIV-1 are members of the viroporin family of small viral membrane proteins. It is essential to determine their structures in order to obtain an understanding of their molecular mechanisms and to develop new classes of anti-viral drugs. Because they are membrane proteins, it is challenging to study them in their native phospholipid bilayer environments by most experimental methods. Here we describe applications of NMR spectroscopy to both p7 and Vpu. Isotopically labeled p7 and Vpu samples were prepared by heterologous expression in bacteria, initial isolation as fusion proteins, and final purification by chromatography. The purified proteins were studied in the model membrane environments of micelles by solution NMR spectroscopy and in aligned phospholipid bilayers by solid-state NMR spectroscopy. The resulting structural findings enable comparisons to be made between the two proteins, demonstrating that they have quite different architectures. Most notably, Vpu has one trans-membrane helix and p7 has two trans-membrane helices; in addition, there are significant differences in the structures and dynamics of their internal loop and terminal regions.► It is feasible to obtain high resolution NMR spectra of p7 and Vpu in both micelle and bilayer environments. ► Both p7 and Vpu have complex architectures with both highly structured and mobile regions. ► p7 and Vpu have different architectures, since p7 has two trans-membrane helices and Vpu has one trans-membrane helix. There are other differences as well.
Keywords: Hepatitis C virus; p7; Membrane protein; Vpu; Human immunodeficiency virus; Nuclear magnetic resonance
Viral channel forming proteins — Modeling the target by Wolfgang B. Fischer; Hao-Jen Hsu (pp. 561-571).
The cellular and subcellular membranes encounter an important playground for the activity of membrane proteins encoded by viruses. Viral membrane proteins, similar to their host companions, can be integral or attached to the membrane. They are involved in directing the cellular and viral reproduction, the fusion and budding processes. This review focuses especially on those integral viral membrane proteins which form channels or pores, the classification to be so, modeling by in silico methods and potential drug candidates. The sequence of an isolate of Vpu from HIV-1 is aligned with host ion channels and a toxin. The focus is on the alignment of the transmembrane domains. The results of the alignment are mapped onto the 3D structures of the respective channels and toxin. The results of the mapping support the idea of a ‘channel–pore dualism’ for Vpu.
Keywords: Viral channel protein; Assembly; Docking; Sequence alignment; Ion channel; Toxin
Viral channel forming proteins — Modeling the target by Wolfgang B. Fischer; Hao-Jen Hsu (pp. 561-571).
The cellular and subcellular membranes encounter an important playground for the activity of membrane proteins encoded by viruses. Viral membrane proteins, similar to their host companions, can be integral or attached to the membrane. They are involved in directing the cellular and viral reproduction, the fusion and budding processes. This review focuses especially on those integral viral membrane proteins which form channels or pores, the classification to be so, modeling by in silico methods and potential drug candidates. The sequence of an isolate of Vpu from HIV-1 is aligned with host ion channels and a toxin. The focus is on the alignment of the transmembrane domains. The results of the alignment are mapped onto the 3D structures of the respective channels and toxin. The results of the mapping support the idea of a ‘channel–pore dualism’ for Vpu.
Keywords: Viral channel protein; Assembly; Docking; Sequence alignment; Ion channel; Toxin
ORF8a of SARS-CoV forms an ion channel: Experiments and molecular dynamics simulations by Cheng-Chang Chen; Kruger Jens Krüger; Issara Sramala; Hao-Jen Hsu; Peter Henklein; Yi-Ming Arthur Chen; Wolfgang B. Fischer (pp. 572-579).
ORF8a protein is 39 residues long and contains a single transmembrane domain. The protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers that forms cation-selective ion channels with a main conductance level of 8.9±0.8pS at elevated temperature (38.5°C). Computational modeling studies including multi nanosecond molecular dynamics simulations in a hydrated POPC lipid bilayer are done with a 22 amino acid transmembrane helix to predict a putative homooligomeric helical bundle model. A structural model of a pentameric bundle is proposed with cysteines, serines and threonines facing the pore.► ORF 8a (39 amino acids) of SARS-CoV forms an ion channel in artificial bilayers. ► Slight cation-selectivity with a main conductance level of 8.9 ± 0.8 pS at elevated temperature. ► Using computational modeling a pentameric assembly is proposed. ► Computational models propose cysteines, serines and threonines facing the pore.
Keywords: SARS-CoV; ORF8a; Ion channel; Molecular dynamics; Bilayer recording
ORF8a of SARS-CoV forms an ion channel: Experiments and molecular dynamics simulations by Cheng-Chang Chen; Kruger Jens Krüger; Issara Sramala; Hao-Jen Hsu; Peter Henklein; Yi-Ming Arthur Chen; Wolfgang B. Fischer (pp. 572-579).
ORF8a protein is 39 residues long and contains a single transmembrane domain. The protein is synthesized using solid phase peptide synthesis and reconstituted into artificial lipid bilayers that forms cation-selective ion channels with a main conductance level of 8.9±0.8pS at elevated temperature (38.5°C). Computational modeling studies including multi nanosecond molecular dynamics simulations in a hydrated POPC lipid bilayer are done with a 22 amino acid transmembrane helix to predict a putative homooligomeric helical bundle model. A structural model of a pentameric bundle is proposed with cysteines, serines and threonines facing the pore.► ORF 8a (39 amino acids) of SARS-CoV forms an ion channel in artificial bilayers. ► Slight cation-selectivity with a main conductance level of 8.9 ± 0.8 pS at elevated temperature. ► Using computational modeling a pentameric assembly is proposed. ► Computational models propose cysteines, serines and threonines facing the pore.
Keywords: SARS-CoV; ORF8a; Ion channel; Molecular dynamics; Bilayer recording
Minimal art: Or why small viral K+ channels are good tools for understanding basic structure and function relations by Gerhard Thiel; Dirk Baumeister; Indra Schroeder; Stefan M. Kast; James L. Van Etten; Anna Moroni (pp. 580-588).
Some algal viruses contain genes that encode proteins with the hallmarks of K+ channels. One feature of these proteins is that they are less than 100 amino acids in size, which make them truly minimal for a K+ channel protein. That is, they consist of only the pore module present in more complex K+ channels. The combination of miniature size and the functional robustness of the viral K+ channels make them ideal model systems for studying how K+ channels work. Here we summarize recent structure/function correlates from these channels, which provide insight into functional properties such as gating, pharmacology and sorting in cells.
Keywords: Kcv; Chlorella virus; Viral ion channel; K; +; channel structure function
Minimal art: Or why small viral K+ channels are good tools for understanding basic structure and function relations by Gerhard Thiel; Dirk Baumeister; Indra Schroeder; Stefan M. Kast; James L. Van Etten; Anna Moroni (pp. 580-588).
Some algal viruses contain genes that encode proteins with the hallmarks of K+ channels. One feature of these proteins is that they are less than 100 amino acids in size, which make them truly minimal for a K+ channel protein. That is, they consist of only the pore module present in more complex K+ channels. The combination of miniature size and the functional robustness of the viral K+ channels make them ideal model systems for studying how K+ channels work. Here we summarize recent structure/function correlates from these channels, which provide insight into functional properties such as gating, pharmacology and sorting in cells.
Keywords: Kcv; Chlorella virus; Viral ion channel; K; +; channel structure function