scholarly journals Control of membrane barrier during bacterial type-III protein secretion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Svenja Hüsing ◽  
Manuel Halte ◽  
Ulf van Look ◽  
Alina Guse ◽  
Eric J. C. Gálvez ◽  
...  
Keyword(s):  
Conformational Changes ◽  
High Speed ◽  
Membrane Integrity ◽  
Salt Bridge ◽  
High Rate ◽  
Salt Bridges ◽  
Secretion Systems ◽  
Bacterial Flagellum ◽  
Type Iii ◽  
Membrane Barrier

AbstractType-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.

scholarly journals Controlling membrane barrier during bacterial type-III protein secretion

2020 ◽  
Author(s):  
Svenja Hüsing ◽  
Ulf van Look ◽  
Alina Guse ◽  
Eric J. C. Gálvez ◽  
Emmanuelle Charpentier ◽  
...  
Keyword(s):  
Conformational Changes ◽  
High Speed ◽  
Membrane Integrity ◽  
Salt Bridge ◽  
High Rate ◽  
Salt Bridges ◽  
Secretion Systems ◽  
Bacterial Flagellum ◽  
Type Iii ◽  
Membrane Barrier

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigated how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.

scholarly journals Injectisome T3SS Subunits As Potential Chaperones In The Extracellular Export of Pectobacterium Carotovorum Subsp. Carotovorum Bacteriocins Carocin S1 And Carocin S3 Secreted Via Flagellar T3SS

2021 ◽  
Author(s):  
Hang-Cheng Chen ◽  
Reymund C. Derilo ◽  
Han-Ling Chen ◽  
Tzu-Rung Li ◽  
Ruchi Briam James S. Lagitnay ◽  
...  
Keyword(s):  
Type Iii Secretion ◽  
Insertional Mutagenesis ◽  
Plant Pathogens ◽  
Soft Rot ◽  
Economic Losses ◽  
Pectobacterium Carotovorum ◽  
Secretion Systems ◽  
Unique Type ◽  
Bacterial Flagellum ◽  
Type Iii

Abstract Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft-rot disease in a wide variety of plants resulting in economic losses worldwide. It produces various types of bacteriocin to compete against related plant pathogens. Studies on how bacteriocins are extracellularly secreted are conducted to understand the mechanism of interbacterial competition. In this study, the secretion of the low-molecular-weight bacteriocins (LMWB) Carocin S1 and Carocin S3 produced by a multiple-bacteriocin producing strain of Pcc, 89-H-4, was investigated. Tn5 insertional mutagenesis was used to generate a mutant, TH22-6, incapable of LMWBs secretion. Sequence and homology analyses of the gene disrupted by transposon Tn5 insertion revealed that the gene sctT, an essential component of the injectisome type III secretion machinery (T3aSS), is required for the secretion of the bacteriocins. This result raised a question regarding the nature of the secretion mechanism of Pcc bacteriocins which was previously discovered to be secreted via T3bSS, a system that utilizes the bacterial flagellum for extracellular secretions. Our previous report has shown that bacteriocin Carocin S1 cannot be secreted by mutants that are defective of T3bSS-related genes such as flhA, flhC, flhD and fliC. We knocked out several genes making up the significant structural components of both T3aSS and T3bSS. The findings led us to hypothesize the potential roles of the T3aSS-related proteins, SctT, SctU and SctV, as flagellar T3SS chaperones in the secretion of Pcc bacteriocins. This current discovery and the findings of our previous study helped us to conceptualize a unique Type III secretion system for bacteriocin extracellular export which is a hybrid of the injectisome and flagellar secretion systems.

scholarly journals Investigating the opening mechanism of topoisomerase I B in complex with DNA by means of metadynamics

2016 ◽  
Author(s):  
Blasco Morozzo della Rocca ◽  
Andrea Coletta ◽  
Federico Iacovelli ◽  
Alessandro Desideri
Keyword(s):  
Conformational Changes ◽  
Topoisomerase I ◽  
Specific Activity ◽  
Dimensional Space ◽  
Center Of Mass ◽  
Minimum Energy ◽  
Salt Bridge ◽  
Computational Techniques ◽  
Salt Bridges ◽  
Collective Variables

Motivations: Topoisomerases play a central role in DNA homeostasis and act on them through a catalytic cycle that comprises the opening of the protein clamp to embrace the DNA (at the beginning of the cycle) and opening to allow the release of the topologically relaxed substrate. Protein opening is hard to follow experimentally thus computational techniques are very handy in dealing with this transitions, although classical molecular dynamics is limited in following big conformational changes and slowly occurring transitions. Therefore we present a well-tempered Metadynamics study of hToP1 clamp opening in presence of a 22-bp long ds-DNA substrate. Methods: The protein structure of wild type hTopIB in complex with DNA has been obtained as previously reported. The hTopIB clamp around the DNA molecule has been destabilized by means of metadynamics using Gromacs-4.5.5 with the PLUMEDv1.3 patch. In the simulation, two Collective Variables have been used to describe the clamp opening: the distance between the center of mass of Cα atoms of the lip1 and of the lip2; and the number of hydrogen bonds between the protein and DNA.Free-energy surface (FES) and the minimum energy path (MEP) connecting different minima were reconstructed using in-house written codes in Python. Salt bridge analysis was conducted via Salt bridges extension of the VMD program and other analyses were performed with the tools of the GROMACS package. Results: Results indicate that the lobes of the protein retain their domain structure during the opening that is characterized by an energy barrier of about 50 Kjoul/mole. Furthermore the DNA remains in contact with the Cap lobe, a fact that would enable the protein to perform 1D-diffusion in the DNA strand to enhance the specific activity by reducing the dimensional space it has to search between relaxation events.

scholarly journals Molecular and cell biological analysis of SwrB in Bacillus subtilis

2021 ◽  
Author(s):  
Andrew M. Phillips ◽  
Sandra Sanchez ◽  
Tatyana A. Sysoeva ◽  
Briana M. Burton ◽  
Daniel B. Kearns
Keyword(s):  
Bacillus Subtilis ◽  
Type Iii Secretion ◽  
Random Mutagenesis ◽  
Basal Bodies ◽  
Secretion Systems ◽  
Swarming Motility ◽  
Bacterial Flagellum ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Critical Residues

Swarming motility is flagellar-mediated movement over a solid surface and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion but the mechanism by which it might perform this function is unknown. Here we show that SwrB likely acts sub-stoichiometrically as it localizes as puncta at the membrane in numbers fewer than that of flagellar basal bodies. Moreover the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely co-localized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs. IMPORTANCE Type III secretion systems (T3SS) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moreover, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here we explore a poorly understood activator of the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.

scholarly journals Salt bridges gate α-catenin activation at intercellular junctions

2018 ◽  
Vol 29 (2) ◽  
pp. 111-122 ◽  
Author(s):  
Samantha Barrick ◽  
Jing Li ◽  
Xinyu Kong ◽  
Alokananda Ray ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Mechanical Activation ◽  
Conformational Changes ◽  
Intercellular Junctions ◽  
Salt Bridge ◽  
Salt Bridges ◽  
Equilibrium Binding ◽  
Binding Data ◽  
Dynamics Simulations ◽  
Intercellular Adhesions

Molecular dynamics simulations, equilibrium binding measurements, and fluorescence imaging reveal the influence of a key salt bridge in the mechanical activation of α-catenin at intercellular adhesions. Simulations reveal possible α-catenin conformational changes underlying experimental fluorescence and equilibrium binding data.

scholarly journals Molecular and cell biological analysis of SwrB in Bacillus subtilis

2021 ◽  
Author(s):  
Andrew M. Phillips ◽  
Sandra Sanchez ◽  
Tatyana A. Sysoeva ◽  
Briana M. Burton ◽  
Daniel B. Kearns
Keyword(s):  
Bacillus Subtilis ◽  
Type Iii Secretion ◽  
Random Mutagenesis ◽  
Basal Bodies ◽  
Secretion Systems ◽  
Swarming Motility ◽  
Bacterial Flagellum ◽  
Type Iii ◽  
Type Iii Secretion Systems ◽  
Critical Residues

ABSTRACTSwarming motility is flagellar-mediated movement over a solid surface and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion but the mechanism by which it might perform this function is unknown. Here we show that SwrB likely acts sub-stoichiometrically as it localizes as puncta at the membrane in numbers fewer than that of flagellar basal bodies. Moreover the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely co-localized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs.IMPORTANCEType III secretion systems (T3SS) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moroever, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here we explore a poorly understood activator the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.

scholarly journals Access to the Complement Factor B Scissile Bond Is Facilitated by Association of Factor B with C3b Protein

2011 ◽  
Vol 286 (41) ◽  
pp. 35725-35732 ◽  
Author(s):  
Dennis E. Hourcade ◽  
Lynne M. Mitchell
Keyword(s):  
Conformational Changes ◽  
Metal Ion ◽  
Alternative Pathway ◽  
Salt Bridge ◽  
Single Amino Acid ◽  
Complement Factor ◽  
Salt Bridges ◽  
Complement Alternative Pathway ◽  
Factor B ◽  
Scissile Bond

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway C3 convertase. During convertase assembly, factor B associates with C3b and Mg2+ forming a pro-convertase C3bB(Mg2+) that is cleaved at a single factor B site by factor D. In free factor B, a pair of salt bridges binds the Arg234 side chain to Glu446 and to Glu207, forming a double latch structure that sequesters the scissile bond (between Arg234 and Lys235) and minimizes its unproductive cleavage. It is unknown how the double latch is released in the pro-convertase. Here, we introduce single amino acid substitutions into factor B that preclude one or both of the Arg234 salt bridges, and we examine their impact on several different pro-convertase complexes. Our results indicate that loss of the Arg234-Glu446 salt bridge partially stabilizes C3bB(Mg2+). Loss of the Arg234-Glu207 salt bridge has lesser effects. We propose that when factor B first associates with C3b, it bears two intact Arg234 salt bridges. The complex rapidly dissociates unless the Arg234-Glu446 salt bridge is released whereupon conformational changes occur that activate the metal ion-dependent adhesion site and partially stabilize the complex. The remaining salt bridge is then released, exposing the scissile bond and permitting factor D cleavage.

scholarly journals Type III secretion systems: the bacterial flagellum and the injectisome

2015 ◽  
Vol 370 (1679) ◽  
pp. 20150020 ◽  
Author(s):  
Andreas Diepold ◽  
Judith P. Armitage
Keyword(s):  
Type Iii Secretion ◽  
Current Knowledge ◽  
Electron Tomography ◽  
Secretion Systems ◽  
Bacterial Flagellum ◽  
Type Iii ◽  
New Findings ◽  
Potential Applications ◽  
Flagellar Filament

The flagellum and the injectisome are two of the most complex and fascinating bacterial nanomachines. At their core, they share a type III secretion system (T3SS), a transmembrane export complex that forms the extracellular appendages, the flagellar filament and the injectisome needle. Recent advances, combining structural biology, cryo-electron tomography, molecular genetics, in vivo imaging, bioinformatics and biophysics, have greatly increased our understanding of the T3SS, especially the structure of its transmembrane and cytosolic components, the transcriptional, post-transcriptional and functional regulation and the remarkable adaptivity of the system. This review aims to integrate these new findings into our current knowledge of the evolution, function, regulation and dynamics of the T3SS, and to highlight commonalities and differences between the two systems, as well as their potential applications.

scholarly journals Investigating the opening mechanism of topoisomerase I B in complex with DNA by means of metadynamics

2016 ◽  
Author(s):  
Blasco Morozzo della Rocca ◽  
Andrea Coletta ◽  
Federico Iacovelli ◽  
Alessandro Desideri
Keyword(s):  
Conformational Changes ◽  
Topoisomerase I ◽  
Specific Activity ◽  
Dimensional Space ◽  
Center Of Mass ◽  
Minimum Energy ◽  
Salt Bridge ◽  
Computational Techniques ◽  
Salt Bridges ◽  
Collective Variables

Motivations: Topoisomerases play a central role in DNA homeostasis and act on them through a catalytic cycle that comprises the opening of the protein clamp to embrace the DNA (at the beginning of the cycle) and opening to allow the release of the topologically relaxed substrate. Protein opening is hard to follow experimentally thus computational techniques are very handy in dealing with this transitions, although classical molecular dynamics is limited in following big conformational changes and slowly occurring transitions. Therefore we present a well-tempered Metadynamics study of hToP1 clamp opening in presence of a 22-bp long ds-DNA substrate. Methods: The protein structure of wild type hTopIB in complex with DNA has been obtained as previously reported. The hTopIB clamp around the DNA molecule has been destabilized by means of metadynamics using Gromacs-4.5.5 with the PLUMEDv1.3 patch. In the simulation, two Collective Variables have been used to describe the clamp opening: the distance between the center of mass of Cα atoms of the lip1 and of the lip2; and the number of hydrogen bonds between the protein and DNA.Free-energy surface (FES) and the minimum energy path (MEP) connecting different minima were reconstructed using in-house written codes in Python. Salt bridge analysis was conducted via Salt bridges extension of the VMD program and other analyses were performed with the tools of the GROMACS package. Results: Results indicate that the lobes of the protein retain their domain structure during the opening that is characterized by an energy barrier of about 50 Kjoul/mole. Furthermore the DNA remains in contact with the Cap lobe, a fact that would enable the protein to perform 1D-diffusion in the DNA strand to enhance the specific activity by reducing the dimensional space it has to search between relaxation events.

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