scholarly journals Sodium-powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan-binding region

2018 ◽  
Author(s):  
Tsubasa Ishida ◽  
Rie Ito ◽  
Jessica Clark ◽  
Nicholas J Matzke ◽  
Yoshiyuki Sowa ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Single Point ◽  
Point Mutations ◽  
Ion Source ◽  
Channel Blockers ◽  
Flagellar Motor ◽  
Binding Region ◽  
Ion Channel Blockers ◽  
Flagellar Motors ◽  
Bacterial Flagellar Motor

SummaryThe bacterial flagellar motor (BFM) powers the rotation that propels swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus / Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium-channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: 1) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus, and 2) near the peptidoglycan (PG) binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers.

scholarly journals Sodium‐powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan‐binding region

2019 ◽  
Vol 111 (6) ◽  
pp. 1689-1699 ◽  
Author(s):  
Tsubasa Ishida ◽  
Rie Ito ◽  
Jessica Clark ◽  
Nicholas J. Matzke ◽  
Yoshiyuki Sowa ◽  
...  
Keyword(s):  
Flagellar Motor ◽  
Binding Region ◽  
Bacterial Flagellar Motor

scholarly journals The bacterial flagellar motor and the evolution of molecular machines

2018 ◽  
Vol 40 (2) ◽  
pp. 4-9
Author(s):  
Morgan Beeby
Keyword(s):  
Molecular Evolution ◽  
Molecular Machines ◽  
Flagellar Motor ◽  
Eukaryotic Evolution ◽  
Flagellar Motors ◽  
Bacterial Flagellar Motor ◽  
Life On Earth

Understanding how life on earth evolved is an enduringly fascinating and profound question. Relative to our understanding of eukaryotic evolution, however, our understanding of how the molecular machines underpinning life have evolved is poor. The bacterial flagellar motor, which drives a rotary propeller for motility, offers a fascinating case study to explore this further, and is now revealing recurring themes in molecular evolution. This article describes recent discoveries about how flagellar motors have diversified since the first flagellar motor evolved, and what this diversity tells us about molecular evolution.

Abstract 431: Probing the Pathogenic Mechanisms Underlying Ca V 1.2 Channelopathies

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Moradeke Bamgboye ◽  
Maria Traficante ◽  
Kevin Herold ◽  
Josiah Owoyemi ◽  
Ivy Dick
Keyword(s):  
Cardiac Function ◽  
Single Point ◽  
Point Mutations ◽  
Electrical Signal ◽  
Channel Blockers ◽  
Phenotypic Variance ◽  
Multisystem Disorder ◽  
Channel Activation ◽  
Cardiac Symptoms ◽  
Dependent Inactivation

The Ca V 1.2 channel is essential to cardiac function. It is the sentinel that translates the electrical signal on the surface of the cell to the intracellular calcium cascade that leads to contraction. To allow the precise tuning of that is essential for cardiac function, Ca V 1.2 is subject to two forms of feedback regulation, voltage dependent inactivation (VDI) and calcium dependent inactivation (CDI). The first mutations described in Ca V 1.2 were causative of Timothy Syndrome (TS), a multisystem disorder with neurological, developmental and life-threatening cardiac symptoms, defined as long-QT type 8 (LQT8). These single point mutations occur in the S6, a region commonly associated with channel activation. In fact, the original TS mutations were shown to perturb channel activation as well as inactivation. Since this first description of TS, multiple additional mutations have been discovered in Ca V 1.2 and are associated with either TS-like or cardiac specific phenotypes. This phenotypic variance suggests that these mutations disrupt channel mechanisms in divergent ways. Moreover, for patients harboring mutations in Ca V 1.2, the cardiac symptoms have proven recalcitrant to conventional therapy including calcium channel blockers (CCBs). This therapeutic inadequacy reinforces the need for a better understanding of how mutations in the S6 disrupt channel function and how this affects therapeutic options for patients that carry them. Here we show that LQT8 mutations that cluster in the S6 of Ca V 1.2 do indeed have a variety of mechanistic underpinnings. This may account for the gradient of symptoms seen in patients with these mutations. In addition, we demonstrate that many of these mutant channels are resistant to block by CCBs, and this resistance strongly correlates to specific mechanistic perturbations of the channel. Thus, reliance on a CCB based therapeutic strategy is likely to leave many in a growing population of patients without a viable treatment option.

scholarly journals Characterization of the Periplasmic Domain of MotB and Implications for Its Role in the Stator Assembly of the Bacterial Flagellar Motor

2008 ◽  
Vol 190 (9) ◽  
pp. 3314-3322 ◽  
Author(s):  
Seiji Kojima ◽  
Yukio Furukawa ◽  
Hideyuki Matsunami ◽  
Tohru Minamino ◽  
Keiichi Namba
Keyword(s):  
Point Mutations ◽  
Nitrilotriacetic Acid ◽  
Inhibitory Effect ◽  
Proton Channel ◽  
Flagellar Motor ◽  
Wild Type ◽  
Deletion Variant ◽  
Periplasmic Domain ◽  
Bacterial Flagellar Motor ◽  
Motility Inhibition

ABSTRACT MotA and MotB are integral membrane proteins that form the stator complex of the proton-driven bacterial flagellar motor. The stator complex functions as a proton channel and couples proton flow with torque generation. The stator must be anchored to an appropriate place on the motor, and this is believed to occur through a putative peptidoglycan-binding (PGB) motif within the C-terminal periplasmic domain of MotB. In this study, we constructed and characterized an N-terminally truncated variant of Salmonella enterica serovar Typhimurium MotB consisting of residues 78 through 309 (MotBC). MotBC significantly inhibited the motility of wild-type cells when exported into the periplasm. Some point mutations in the PGB motif enhanced the motility inhibition, while an in-frame deletion variant, MotBC(Δ197-210), showed a significantly reduced inhibitory effect. Wild-type MotBC and its point mutant variants formed a stable homodimer, while the deletion variant was monomeric. A small amount of MotB was coisolated only with the secreted form of MotBC-His6 by Ni-nitrilotriacetic acid affinity chromatography, suggesting that the motility inhibition results from MotB-MotBC heterodimer formation in the periplasm. However, the monomeric mutant variant MotBC(Δ197-210) did not bind to MotB, suggesting that MotBC is directly involved in stator assembly. We propose that the MotBC dimer domain plays an important role in targeting and stable anchoring of the MotA/MotB complex to putative stator-binding sites of the motor.

scholarly journals A single lysine in the N-terminal region of store-operated channels is critical for STIM1-mediated gating

2010 ◽  
Vol 136 (6) ◽  
pp. 673-686 ◽  
Author(s):  
Annette Lis ◽  
Susanna Zierler ◽  
Christine Peinelt ◽  
Andrea Fleig ◽  
Reinhold Penner
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Calcium Release ◽  
Transmembrane Domain ◽  
Single Point ◽  
Point Mutations ◽  
Terminal Region ◽  
Single Amino Acid ◽  
Critical Element ◽  
Crac Channels

Store-operated Ca2+ entry is controlled by the interaction of stromal interaction molecules (STIMs) acting as endoplasmic reticulum ER Ca2+ sensors with calcium release–activated calcium (CRAC) channels (CRACM1/2/3 or Orai1/2/3) in the plasma membrane. Here, we report structural requirements of STIM1-mediated activation of CRACM1 and CRACM3 using truncations, point mutations, and CRACM1/CRACM3 chimeras. In accordance with previous studies, truncating the N-terminal region of CRACM1 or CRACM3 revealed a 20–amino acid stretch close to the plasma membrane important for channel gating. Exchanging the N-terminal region of CRACM3 with that of CRACM1 (CRACM3-N(M1)) results in accelerated kinetics and enhanced current amplitudes. Conversely, transplanting the N-terminal region of CRACM3 into CRACM1 (CRACM1-N(M3)) leads to severely reduced store-operated currents. Highly conserved amino acids (K85 in CRACM1 and K60 in CRACM3) in the N-terminal region close to the first transmembrane domain are crucial for STIM1-dependent gating of CRAC channels. Single-point mutations of this residue (K85E and K60E) eliminate store-operated currents induced by inositol 1,4,5-trisphosphate and reduce store-independent gating by 2-aminoethoxydiphenyl borate. However, short fragments of these mutant channels are still able to communicate with the CRAC-activating domain of STIM1. Collectively, these findings identify a single amino acid in the N terminus of CRAC channels as a critical element for store-operated gating of CRAC channels.

scholarly journals The Response of Bacterial Flagellar Motor to Stepwise Increase in NaCl Concentration

2020 ◽  
Author(s):  
V. Soman ◽  
S. Kumari ◽  
S. Nath ◽  
R. Elangovan
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Salmonella Enteritidis ◽  
Proton Motive Force ◽  
Motive Force ◽  
Flagellar Motor ◽  
Stepwise Increase ◽  
Flagellar Motors ◽  
Nacl Concentration ◽  
Bacterial Flagellar Motor

AbstractMany species of bacteria use flagella to navigate in its environment. The flagellum is a 7-10 μm long helical filament with a rotary motor at its base embedded in the cell membrane and almost a dozen stator complexes. Proton motive force across the cell membrane powers the flagellar motors of E.coli and Salmonella. The motor stochastically switches between clockwise and counter-clockwise direction. A chemotaxis system causes the motor to change its direction, but the process is more complex as the switch is sensitive to load and proton motive force as well. NaCl is significant with regard to the flagellar motor as it affects the stator dynamics, proton motive force, and osmotaxis at higher concentration. Chemotaxis helps the bacteria for its growth and survival. E.coli’s natural habitat has high osmolarity and the organism uses use various mechanisms for osmoregulation. However, the role of flagellar motor to adapt to the changes in osmolarity, or osmotaxis, is not well studied. In this work, we dissipated the membrane potential of bacteria in pH 7 using step-wise increase in concentration of NaCl in motility buffer and studied the output of E.coli’s flagellar motor using tethered bead assay and swimming Salmonella enteritidis cells. We observed decrease in motor speed and switching rates with stepwise increase in NaCl concentration in the motility buffer. The mean speed of the motors decreased with NaCl concentration. The population of swimming cells tumbled more with increase in concentration of NaCl. At the single motor level, the motors biased to CCW rotation with decrease in membrane potential. In this study, we present our observations of the flagellar motor in high NaCl concentration, and explore how NaCl can be used to study various aspects of the bacterial flagellar motor.Statement of significanceSodium ion has been significant in the both the cellular energetics and the function of bacterial flagellar motor. Growing evidence show that the effect of sodium ions was not what hitherto thought it would be. It is involved in the sodium energetics, dissipate membrane potential, affect the flagellar stator dynamics of bacteria. Being an osmolyte, it influences the osmotaxis of bacteria. In this work, we studied the effect of NaCl on the response of the single bacterial flagellar motor of E.coli and swimming cells of Salmonella enteritidis. We observed that the effect of NaCl on the output of the flagellar motor was significant and it may affect the cells in various ways.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

2021 ◽  
Author(s):  
Marisa L. Martino ◽  
Stephen N. Crooke ◽  
Marianne Manchester ◽  
M.G. Finn
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Virus Like Particles ◽  
Single Point Mutations

scholarly journals mCSM-membrane: predicting the effects of mutations on transmembrane proteins

2020 ◽  
Vol 48 (W1) ◽  
pp. W147-W153 ◽  
Author(s):  
Douglas E V Pires ◽  
Carlos H M Rodrigues ◽  
David B Ascher
Keyword(s):  
Membrane Proteins ◽  
Single Point ◽  
Point Mutations ◽  
Web Server ◽  
Protein Coding ◽  
Pathogenic Variants ◽  
Model Protein ◽  
Matthew’S Correlation Coefficient ◽  
Membrane Protein Stability ◽  
User Friendly

Abstract Significant efforts have been invested into understanding and predicting the molecular consequences of mutations in protein coding regions, however nearly all approaches have been developed using globular, soluble proteins. These methods have been shown to poorly translate to studying the effects of mutations in membrane proteins. To fill this gap, here we report, mCSM-membrane, a user-friendly web server that can be used to analyse the impacts of mutations on membrane protein stability and the likelihood of them being disease associated. mCSM-membrane derives from our well-established mutation modelling approach that uses graph-based signatures to model protein geometry and physicochemical properties for supervised learning. Our stability predictor achieved correlations of up to 0.72 and 0.67 (on cross validation and blind tests, respectively), while our pathogenicity predictor achieved a Matthew's Correlation Coefficient (MCC) of up to 0.77 and 0.73, outperforming previously described methods in both predicting changes in stability and in identifying pathogenic variants. mCSM-membrane will be an invaluable and dedicated resource for investigating the effects of single-point mutations on membrane proteins through a freely available, user friendly web server at http://biosig.unimelb.edu.au/mcsm_membrane.

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