Journal Articles
Functional Monomerization of a ClC-Type Fluoride Transporter
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22
Author(s): Nicholas B. Last, Christopher Miller
Anion channels and antiporters of the ClC superfamily have been found to be exclusively dimeric in nature, even though each individual monomer contains the complete transport pathway. Here, we describe the destabilization through mutagenesis of the dimer interface of a bacterial F−/H+ antiporter, ClCF-eca. Several mutations that produce monomer/dimer equilibrium of the normally dimeric transporter were found, simply by shortening a hydrophobic side chain in some cases. One mutation, L376W, leads to a wholly monomeric variant that shows full activity. Furthermore, we discovered a naturally destabilized homologue, ClCF-rla, which undergoes partial monomerization in detergent without additional mutations. These results, in combination with the previous functional monomerization of the distant relative ClC-ec1, demonstrate that the monomer alone is the functional unit for several clades of the ClC superfamily.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 22
Author(s): Nicholas B. Last, Christopher Miller
Anion channels and antiporters of the ClC superfamily have been found to be exclusively dimeric in nature, even though each individual monomer contains the complete transport pathway. Here, we describe the destabilization through mutagenesis of the dimer interface of a bacterial F−/H+ antiporter, ClCF-eca. Several mutations that produce monomer/dimer equilibrium of the normally dimeric transporter were found, simply by shortening a hydrophobic side chain in some cases. One mutation, L376W, leads to a wholly monomeric variant that shows full activity. Furthermore, we discovered a naturally destabilized homologue, ClCF-rla, which undergoes partial monomerization in detergent without additional mutations. These results, in combination with the previous functional monomerization of the distant relative ClC-ec1, demonstrate that the monomer alone is the functional unit for several clades of the ClC superfamily.
Graphical abstract
Categories: Journal Articles
Protein–Protein Interfaces in Viral Capsids Are Structurally Unique
Publication date: 6 November 2015
Source:Journal of Molecular Biology, Volume 427, Issue 22
Author(s): Shanshan Cheng, Charles L. Brooks
Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein–protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein–protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein–protein interfaces that share similar physicochemical patterns with some protein–protein interfaces in the capsid set, using the program PCalign we developed for comparing protein–protein interfaces. This overlap in the structural space of protein–protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein–protein interfaces. Furthermore, the generic protein–protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein–protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 22
Author(s): Shanshan Cheng, Charles L. Brooks
Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein–protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein–protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein–protein interfaces that share similar physicochemical patterns with some protein–protein interfaces in the capsid set, using the program PCalign we developed for comparing protein–protein interfaces. This overlap in the structural space of protein–protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein–protein interfaces. Furthermore, the generic protein–protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein–protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy.
Graphical abstract
Categories: Journal Articles
Conformational Transitions that Enable Histidine Kinase Autophosphorylation and Receptor Array Integration
Publication date: Available online 30 October 2015
Source:Journal of Molecular Biology
Author(s): Anna R. Greenswag, Alise Muok, Xiaoxiao Li, Brian R. Crane
During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric, histidine-kinase CheA. The five domains of CheA (P1-P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and x-ray scattering studies of thermostable CheA from Thermotoga maritima find that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP nor ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains, or removal of one within a dimer, increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates inter-subunit (trans) versus intra-subunit (cis) autophosphorylation; with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans-directing interactions between the tether connecting P3 to P2-P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and suggests that the ATP-lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Anna R. Greenswag, Alise Muok, Xiaoxiao Li, Brian R. Crane
During bacterial chemotaxis, transmembrane chemoreceptor arrays regulate autophosphorylation of the dimeric, histidine-kinase CheA. The five domains of CheA (P1-P5) each play a specific role in coupling receptor stimulation to CheA activity. Biochemical and x-ray scattering studies of thermostable CheA from Thermotoga maritima find that the His-containing substrate domain (P1) is sequestered by interactions that depend upon P1 of the adjacent subunit. Non-hydrolyzable ATP analogs (but not ATP nor ADP) release P1 from the protein core (domains P3P4P5) and increase its mobility. Detachment of both P1 domains, or removal of one within a dimer, increases net autophosphorylation substantially at physiological temperature (55°C). However, nearly all activity is lost without the dimerization domain (P3). The linker length between P1 and P3 dictates inter-subunit (trans) versus intra-subunit (cis) autophosphorylation; with the trans reaction requiring a minimum length of 47 residues. A new crystal structure of the most active dimerization-plus-kinase unit (P3P4) reveals trans-directing interactions between the tether connecting P3 to P2-P1 and the adjacent ATP-binding (P4) domain. The orientation of P4 relative to P3 in the P3P4 structure supports a planar CheA conformation that is required by membrane array models, and suggests that the ATP-lid of CheA may be poised to interact with receptors and coupling proteins. Collectively, these data suggest that the P1 domains are restrained in the off-state as a result of cross-subunit interactions. Perturbations at the nucleotide-binding pocket increase P1 mobility and access of the substrate His to P4-bound ATP.
Graphical abstract
Categories: Journal Articles
Identification and characterization of botulinum neurotoxin A substrate binding pockets and their re-engineering for human SNAP-23
Publication date: Available online 30 October 2015
Source:Journal of Molecular Biology
Author(s): Stefan Sikorra, Christa Litschko, Carina Müller, Nadine Thiel, Thierry Galli, Timo Eichner, Thomas Binz
Botulinum neurotoxins are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs. However, their serotype A (BoNT/A) which cleaves synaptosomal associated protein of 25 kDa (SNAP-25) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of ten SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23 mediated hypersecretion.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Stefan Sikorra, Christa Litschko, Carina Müller, Nadine Thiel, Thierry Galli, Timo Eichner, Thomas Binz
Botulinum neurotoxins are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs. However, their serotype A (BoNT/A) which cleaves synaptosomal associated protein of 25 kDa (SNAP-25) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of ten SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23 mediated hypersecretion.
Graphical abstract
Categories: Journal Articles
The bacterial transcription termination factor Rho coordinates Mg2+ homeostasis with translational signals
Publication date: Available online 30 October 2015
Source:Journal of Molecular Biology
Author(s): Michelle A. Kriner, Eduardo A. Groisman
The bacterial protein Rho triggers transcription termination at the ends of many operons and when transcription and translation become uncoupled. In addition to these genome-wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5’ leader region or continues into the downstream coding region. Here, we report that the Mg2+ channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5’ leader region. We demonstrate that the unusually long and highly conserved corA leader mRNA can adopt two mutually exclusive conformations that determine whether or not Rho interacts with a Rho utilization (rut) site on the nascent RNA and thereby prevents transcription of the corA coding region. The RNA conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Thus, corA transcription is inversely coupled to corL translation. This mechanism resembles those governing expression of Salmonella’s other two Mg2+ transport genes, suggesting that Rho links Mg2+ uptake to translational signals.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Michelle A. Kriner, Eduardo A. Groisman
The bacterial protein Rho triggers transcription termination at the ends of many operons and when transcription and translation become uncoupled. In addition to these genome-wide activities, Rho implements regulation of specific genes by dictating whether RNA polymerase terminates transcription within the 5’ leader region or continues into the downstream coding region. Here, we report that the Mg2+ channel gene corA in Salmonella enterica serovar Typhimurium, which was previously thought to be constitutively expressed, is regulated by a Rho-dependent terminator located within its 5’ leader region. We demonstrate that the unusually long and highly conserved corA leader mRNA can adopt two mutually exclusive conformations that determine whether or not Rho interacts with a Rho utilization (rut) site on the nascent RNA and thereby prevents transcription of the corA coding region. The RNA conformation that promotes Rho-dependent termination is favored by efficient translation of corL, a short open reading frame located within the corA leader. Thus, corA transcription is inversely coupled to corL translation. This mechanism resembles those governing expression of Salmonella’s other two Mg2+ transport genes, suggesting that Rho links Mg2+ uptake to translational signals.
Graphical abstract
Categories: Journal Articles
Structural biology and regulation of protein import into the nucleus
Publication date: Available online 30 October 2015
Source:Journal of Molecular Biology
Author(s): Mary Christie, Chiung-Wen Chang, Gergely Róna, Kate M. Smith, Alastair G. Stewart, Agnes A.S. Takeda, Marcos R.M. Fontes, Murray Stewart, Beáta G. Vértessy, Jade K. Forwood, Bostjan Kobe
Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g. importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g. importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals (cNLSs), recognized by the adaptor importin-α, and the PY-NLSs, recognized by transportin-1. Post-translational modifications, in particular phosphorylation, constitute key regulatory mechanisms operating in these pathways.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Mary Christie, Chiung-Wen Chang, Gergely Róna, Kate M. Smith, Alastair G. Stewart, Agnes A.S. Takeda, Marcos R.M. Fontes, Murray Stewart, Beáta G. Vértessy, Jade K. Forwood, Bostjan Kobe
Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the β-karyopherin family, which can bind cargo directly (e.g. importin-β, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g. importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals (cNLSs), recognized by the adaptor importin-α, and the PY-NLSs, recognized by transportin-1. Post-translational modifications, in particular phosphorylation, constitute key regulatory mechanisms operating in these pathways.
Graphical abstract
Categories: Journal Articles
Base flipping by MTERF1 can accommodate multiple conformations and occurs in a stepwise fashion
Publication date: Available online 30 October 2015
Source:Journal of Molecular Biology
Author(s): James Byrnes, Kevin Hauser, Leah Norona, Edison Mejia, Carlos Simmerling, Miguel Garcia-Diaz
Human mitochondrial transcription termination occurs within the leu-tRNA gene and is mediated by the DNA binding protein MTERF1. The crystal structure of MTERF1 bound to the canonical termination sequence reveals a rare base flipping event that involves the eversion of three nucleotides. These nucleotides are stabilized by stacking interactions with three MTERF1 residues, which are not only essential for base flipping but also for termination activity. To further understand the mechanism of base flipping we examined each of the individual stacking interactions in structural, energetic and functional detail. Individual substitutions of Arg162, Tyr288 and Phe243 have revealed unequal contributions to overall termination activity. Furthermore, our work identifies an important role for Phe322 in the base flipping mechanism and we demonstrate how Phe322 and Phe243 are important for coupling base flipping between the heavy and light strand DNA chains. We propose a step-wise model for the base flipping process that recapitulates our observations. Finally, we show that MTERF1 has the ability to accommodate alternate active conformations. The adaptability of base flipping has implications for MTERF1 function and for the putative function of MTERF1 at alternative binding sites in human mitochondria.
Source:Journal of Molecular Biology
Author(s): James Byrnes, Kevin Hauser, Leah Norona, Edison Mejia, Carlos Simmerling, Miguel Garcia-Diaz
Human mitochondrial transcription termination occurs within the leu-tRNA gene and is mediated by the DNA binding protein MTERF1. The crystal structure of MTERF1 bound to the canonical termination sequence reveals a rare base flipping event that involves the eversion of three nucleotides. These nucleotides are stabilized by stacking interactions with three MTERF1 residues, which are not only essential for base flipping but also for termination activity. To further understand the mechanism of base flipping we examined each of the individual stacking interactions in structural, energetic and functional detail. Individual substitutions of Arg162, Tyr288 and Phe243 have revealed unequal contributions to overall termination activity. Furthermore, our work identifies an important role for Phe322 in the base flipping mechanism and we demonstrate how Phe322 and Phe243 are important for coupling base flipping between the heavy and light strand DNA chains. We propose a step-wise model for the base flipping process that recapitulates our observations. Finally, we show that MTERF1 has the ability to accommodate alternate active conformations. The adaptability of base flipping has implications for MTERF1 function and for the putative function of MTERF1 at alternative binding sites in human mitochondria.
Categories: Journal Articles
Determination of Protein Folding Intermediate Structures Consistent with Data from Oxidative Footprinting Mass Spectrometry
Publication date: Available online 30 October 2015
Source:Journal of Molecular Biology
Author(s): Florian Heinkel, Jörg Gsponer
The mapping of folding landscapes remains an important challenge in protein chemistry. Pulsed oxidative labeling of exposed residues and their detection via mass spectrometry provides new means of taking time-resolved “snapshots” of the structural changes that occur during protein folding. However, such experiments have so far only been interpreted qualitatively. Here, we report the detailed structural interpretation of mass spectrometry data from fast photochemical oxidation of proteins (FPOP) experiments at atomic resolution in a biased molecular dynamics approach. We are able to calculate structures of the early folding intermediate of the model system barstar that are fully consistent with FPOP data as well as Φ values. Furthermore, structures calculated with both FPOP data and Φ values are significantly less compact and have fewer helical residues than intermediate structures calculated with Φ values only. This improves the agreement with the experimental β-Tanford value as well as CD measurements. The restraints that we introduce facilitate the structural interpretation of FPOP data and provide new means for refined structure calculations of transiently samples states on protein folding landscapes.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Florian Heinkel, Jörg Gsponer
The mapping of folding landscapes remains an important challenge in protein chemistry. Pulsed oxidative labeling of exposed residues and their detection via mass spectrometry provides new means of taking time-resolved “snapshots” of the structural changes that occur during protein folding. However, such experiments have so far only been interpreted qualitatively. Here, we report the detailed structural interpretation of mass spectrometry data from fast photochemical oxidation of proteins (FPOP) experiments at atomic resolution in a biased molecular dynamics approach. We are able to calculate structures of the early folding intermediate of the model system barstar that are fully consistent with FPOP data as well as Φ values. Furthermore, structures calculated with both FPOP data and Φ values are significantly less compact and have fewer helical residues than intermediate structures calculated with Φ values only. This improves the agreement with the experimental β-Tanford value as well as CD measurements. The restraints that we introduce facilitate the structural interpretation of FPOP data and provide new means for refined structure calculations of transiently samples states on protein folding landscapes.
Graphical abstract
Categories: Journal Articles
The kink-turn, a key architectural element in RNA structure
Publication date: Available online 29 October 2015
Source:Journal of Molecular Biology
Author(s): Lin Huang, David M.J. Lilley
Kink-turns (k-turns) are widespread structural elements that introduce an axial bend into duplex RNA with an included angle of 50°. These mediate key tertiary interactions, and bind specific proteins including members of the L7Ae family. The standard k-turn comprises a three-nucleotide bulge followed by G•A and A•G pairs. The RNA kinks by an association of the two minor grooves, stabilized by the formation of a number of key cross-strand hydrogen bonds mostly involving the adenine bases of the G•A and A•G pairs. The k-turns may be divided into two conformational classes, depending on the receptor for one of these hydrogen bonds. k-turns become folded by one of three different processes. Some, but not all, k-turns become folded in the present of metal ions. Whether or not a given k-turn is folded under these conditions is determined by its sequence. We present a set of rules for the prediction of folding properties, and the structure adopted, on local sequence.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Lin Huang, David M.J. Lilley
Kink-turns (k-turns) are widespread structural elements that introduce an axial bend into duplex RNA with an included angle of 50°. These mediate key tertiary interactions, and bind specific proteins including members of the L7Ae family. The standard k-turn comprises a three-nucleotide bulge followed by G•A and A•G pairs. The RNA kinks by an association of the two minor grooves, stabilized by the formation of a number of key cross-strand hydrogen bonds mostly involving the adenine bases of the G•A and A•G pairs. The k-turns may be divided into two conformational classes, depending on the receptor for one of these hydrogen bonds. k-turns become folded by one of three different processes. Some, but not all, k-turns become folded in the present of metal ions. Whether or not a given k-turn is folded under these conditions is determined by its sequence. We present a set of rules for the prediction of folding properties, and the structure adopted, on local sequence.
Graphical abstract
Categories: Journal Articles
Breaching the barrier - the nuclear envelope in virus infection
Publication date: Available online 29 October 2015
Source:Journal of Molecular Biology
Author(s): Thomas C. Mettenleiter
Many DNA and a few RNA viruses use the host cell nucleus for virion formation and/or genome replication. To this end, the nuclear envelope barrier has to be overcome for entry into and egress from the intranuclear replication compartment. Different virus families have devised ingenious ways of entering and leaving the nucleus usurping cellular transport pathways through the nuclear pore complex but also translocating directly through both membranes of the nuclear envelope. This intriguing diversity in nuclear entry and egress of viruses also highlights different ways nucleo-cytoplasmic transport can occur. Thus, the study of interactions between viruses and the nuclear envelope also helps to unravel hitherto unknown cellular pathways such as vesicular nucleo-cytoplasmic transfer.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Thomas C. Mettenleiter
Many DNA and a few RNA viruses use the host cell nucleus for virion formation and/or genome replication. To this end, the nuclear envelope barrier has to be overcome for entry into and egress from the intranuclear replication compartment. Different virus families have devised ingenious ways of entering and leaving the nucleus usurping cellular transport pathways through the nuclear pore complex but also translocating directly through both membranes of the nuclear envelope. This intriguing diversity in nuclear entry and egress of viruses also highlights different ways nucleo-cytoplasmic transport can occur. Thus, the study of interactions between viruses and the nuclear envelope also helps to unravel hitherto unknown cellular pathways such as vesicular nucleo-cytoplasmic transfer.
Graphical abstract
Categories: Journal Articles
Mechanistic insights from structural analyses of Ran GTPase-driven nuclear export of proteins and RNAs
Publication date: Available online 28 October 2015
Source:Journal of Molecular Biology
Author(s): Yoshiyuki Matsuura
Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes (NPCs) is a fundamental problem in biology. Exportins are Ran GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic phenylalanine-glycine (FG) repeat motifs facilitate NPC passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release, and also beginning to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Yoshiyuki Matsuura
Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes (NPCs) is a fundamental problem in biology. Exportins are Ran GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic phenylalanine-glycine (FG) repeat motifs facilitate NPC passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release, and also beginning to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza.
Graphical abstract
Categories: Journal Articles
Intermicrobial interactions as a driver for community composition and stratification of oral biofilms
Publication date: Available online 28 October 2015
Source:Journal of Molecular Biology
Author(s): Nicholas S. Jakubovics
The oral cavity is accessible to microorganisms, and biofilms are present throughout on hard and soft tissues. The shedding of epithelial cell layers is usually effective for controlling biofilm development on soft tissues. Innate immune mechanisms are not so effective against biofilms on tooth surfaces, and oral hygiene measures such as brushing and flossing are required for the periodic removal of dental plaque. Even with good oral hygiene, microbial communities accumulate on teeth in areas that are protected from mechanical abrasion forces. Changes in the composition of these biofilms are associated with oral diseases such as dental caries or periodontitis. Newly formed biofilms and more mature dental plaque each have a level of spatial organization in the horizontal and vertical planes. Communities are shaped by many varied interactions between different species and genera within the biofilm, which include physical cell-cell associations known as coaggregation, interspecies signaling, secretion and turnover of antimicrobial compounds, and the sharing of an extracellular matrix. Central to these interactions is the selection for metabolic synergies and it is becoming clear that the ability of communities to extract the maximum energy from the available metabolites is a potent driver for biofilm structure and stratification. This review discusses recent advances in our understanding of intermicrobial interactions in oral biofilms and the roles that they play in determining the spatial organization of biofilm communities.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Nicholas S. Jakubovics
The oral cavity is accessible to microorganisms, and biofilms are present throughout on hard and soft tissues. The shedding of epithelial cell layers is usually effective for controlling biofilm development on soft tissues. Innate immune mechanisms are not so effective against biofilms on tooth surfaces, and oral hygiene measures such as brushing and flossing are required for the periodic removal of dental plaque. Even with good oral hygiene, microbial communities accumulate on teeth in areas that are protected from mechanical abrasion forces. Changes in the composition of these biofilms are associated with oral diseases such as dental caries or periodontitis. Newly formed biofilms and more mature dental plaque each have a level of spatial organization in the horizontal and vertical planes. Communities are shaped by many varied interactions between different species and genera within the biofilm, which include physical cell-cell associations known as coaggregation, interspecies signaling, secretion and turnover of antimicrobial compounds, and the sharing of an extracellular matrix. Central to these interactions is the selection for metabolic synergies and it is becoming clear that the ability of communities to extract the maximum energy from the available metabolites is a potent driver for biofilm structure and stratification. This review discusses recent advances in our understanding of intermicrobial interactions in oral biofilms and the roles that they play in determining the spatial organization of biofilm communities.
Graphical abstract
Categories: Journal Articles
PhyreStorm: A web server for fast structural searches against the PDB
Publication date: Available online 27 October 2015
Source:Journal of Molecular Biology
Author(s): Stefans Mezulis, Michael J.E. Sternberg, Lawrence A. Kelley
The identification of structurally similar proteins can provide a range of biological insights and accordingly the alignment of a query protein to a database of experimentally-determined protein structures is a technique commonly used in the fields of structural and evolutionary biology. The PhyreStorm web server has been designed to provide comprehensive, up-to-date and rapid structural comparisons against the Protein Data Bank (PDB) combined with a rich and intuitive user interface. It is intended that this facility will enable biologists inexpert in bioinformatics access to a powerful tool for exploring protein structure relationships beyond what can be achieved by sequence analysis alone. By partitioning the PDB into similar structures, PhyreStorm is able to quickly discard the majority of structures that cannot possibly align well to a query protein, reducing the number of alignments required by an order of magnitude. PhyreStorm is capable of finding 93±2% of all highly similar (TM-score >0.7) structures in the PDB for each query structure, usually in under 60 seconds. PhyreStorm is available at http://www.sbg.bio.ic.ac.uk/phyrestorm/
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Stefans Mezulis, Michael J.E. Sternberg, Lawrence A. Kelley
The identification of structurally similar proteins can provide a range of biological insights and accordingly the alignment of a query protein to a database of experimentally-determined protein structures is a technique commonly used in the fields of structural and evolutionary biology. The PhyreStorm web server has been designed to provide comprehensive, up-to-date and rapid structural comparisons against the Protein Data Bank (PDB) combined with a rich and intuitive user interface. It is intended that this facility will enable biologists inexpert in bioinformatics access to a powerful tool for exploring protein structure relationships beyond what can be achieved by sequence analysis alone. By partitioning the PDB into similar structures, PhyreStorm is able to quickly discard the majority of structures that cannot possibly align well to a query protein, reducing the number of alignments required by an order of magnitude. PhyreStorm is capable of finding 93±2% of all highly similar (TM-score >0.7) structures in the PDB for each query structure, usually in under 60 seconds. PhyreStorm is available at http://www.sbg.bio.ic.ac.uk/phyrestorm/
Graphical abstract
Categories: Journal Articles
A Snapshot of the Extraordinary World of Social Microbiology
Publication date: Available online 27 October 2015
Source:Journal of Molecular Biology
Author(s): Nicola R. Stanley-Wall, Sarah J. Coulthurst, Ian Barry Holland
Source:Journal of Molecular Biology
Author(s): Nicola R. Stanley-Wall, Sarah J. Coulthurst, Ian Barry Holland
Categories: Journal Articles
Bacterial Networks in Cells and Communities
Publication date: Available online 24 October 2015
Source:Journal of Molecular Biology
Author(s): Victor Sourjik, Julia A. Vorholt
Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks (BacNet15) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (−omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Victor Sourjik, Julia A. Vorholt
Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks (BacNet15) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (−omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria.
Graphical abstract
Categories: Journal Articles
Editorial Board
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Source:Journal of Molecular Biology, Volume 427, Issue 21
Categories: Journal Articles
Contents List
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Source:Journal of Molecular Biology, Volume 427, Issue 21
Categories: Journal Articles
Omics Approaches Deciphering Molecular Function in Large Biological Systems
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Marina Ostankovitch, Igor Stagljar
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Marina Ostankovitch, Igor Stagljar
Categories: Journal Articles
Using Transcriptional Signatures to Assess Immune Cell Function: From Basic Mechanisms to Immune-Related Disease
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Maxime Touzot, Alix Dahirel, Antonio Cappuccio, Elodie Segura, Philippe Hupé, Vassili Soumelis
Assessing human immune response remains a challenge as it involves multiple cell types in specific tissues. The use of microarray-based expression profiling as a tool for assessing the immune response has grown increasingly over the past decade. Transcriptome analyses provide investigators with a global perspective of the complex molecular and cellular events that unfold during the development of an immune response. In this review, we will detail the broad use of gene expression profiling to decipher the complexity of immune responses from disease biomarkers identification to cell activation, polarisation or functional specialisation. We will also describe how such data-driven strategies revealed the flexibility of immune function with common and specific transcriptional programme under multiple stimuli.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Maxime Touzot, Alix Dahirel, Antonio Cappuccio, Elodie Segura, Philippe Hupé, Vassili Soumelis
Assessing human immune response remains a challenge as it involves multiple cell types in specific tissues. The use of microarray-based expression profiling as a tool for assessing the immune response has grown increasingly over the past decade. Transcriptome analyses provide investigators with a global perspective of the complex molecular and cellular events that unfold during the development of an immune response. In this review, we will detail the broad use of gene expression profiling to decipher the complexity of immune responses from disease biomarkers identification to cell activation, polarisation or functional specialisation. We will also describe how such data-driven strategies revealed the flexibility of immune function with common and specific transcriptional programme under multiple stimuli.
Graphical abstract
Categories: Journal Articles
Measuring Transcription Rate Changes via Time-Course 4-Thiouridine Pulse-Labelling Improves Transcriptional Target Identification
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Thomas Schwarzl, Desmond G. Higgins, Walter Kolch, David J. Duffy
Identifying changes in the transcriptional regulation of target genes from high-throughput studies is important for unravelling molecular mechanisms controlled by a given perturbation. When measuring global transcript levels only, the effect of the perturbation [e.g., transcription factor (TF) overexpression or drug treatment] on its target genes is often obscured by delayed feedback and secondary effects until the changes are fully propagated. As a proof of principle, we show that selective measuring of transcripts that are only synthesised after a perturbation [4-thiouridine (4sU) sequencing (4sU-seq)] is a more sensitive method to identify targets and time-dependent transcriptional responses than global transcript profiling. By metabolically labelling RNA in a time-course setup, we could vastly increase the sensitivity of MYCN target gene detection compared to traditional RNA sequencing. The validity of targets identified by 4sU-seq was demonstrated using chromatin immunoprecipitation sequencing and neuroblastoma microarray tumour data. Here, we describe the methodology, both molecular biology and computational aspects, required to successfully apply this 4sU-seq approach.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Thomas Schwarzl, Desmond G. Higgins, Walter Kolch, David J. Duffy
Identifying changes in the transcriptional regulation of target genes from high-throughput studies is important for unravelling molecular mechanisms controlled by a given perturbation. When measuring global transcript levels only, the effect of the perturbation [e.g., transcription factor (TF) overexpression or drug treatment] on its target genes is often obscured by delayed feedback and secondary effects until the changes are fully propagated. As a proof of principle, we show that selective measuring of transcripts that are only synthesised after a perturbation [4-thiouridine (4sU) sequencing (4sU-seq)] is a more sensitive method to identify targets and time-dependent transcriptional responses than global transcript profiling. By metabolically labelling RNA in a time-course setup, we could vastly increase the sensitivity of MYCN target gene detection compared to traditional RNA sequencing. The validity of targets identified by 4sU-seq was demonstrated using chromatin immunoprecipitation sequencing and neuroblastoma microarray tumour data. Here, we describe the methodology, both molecular biology and computational aspects, required to successfully apply this 4sU-seq approach.
Graphical abstract
Categories: Journal Articles
